Novel transporter constructs and transporter cargo conjugate molecules

ABSTRACT

The present invention relates to novel transporter constructs of the generic formula (I) DlLLLxDm(LLLyDn)a and variants thereof. The present invention also refers to transporter cargo conjugate molecules, particularly of conjugates of the novel transporter constructs with a cargo moiety, e.g. proteins or peptides, nucleic acids, cytotoxic agents, organic molecules, etc. The present invention furthermore discloses (pharmaceutical) compositions comprising these conjugates and methods of treatment and uses involving such transporter constructs.

FIELD

The present invention relates to novel transporter constructs of thegeneric formula (I) D_(l)LLL_(x)D_(m)(LLL_(y)D_(n))_(a) and variantsthereof. The present invention also refers to transporter cargoconjugate molecules, particularly of conjugates of the novel transporterconstructs with a cargo moiety, e.g. proteins or peptides, nucleicacids, cytotoxic agents, organic molecules, etc. The present inventionfurthermore discloses (pharmaceutical) compositions comprising theseconjugates and methods of treatment and uses involving such transporterconstructs.

BACKGROUND

Techniques enabling efficient transfer of a substance of interest fromthe external medium into tissue or cells, and particularly to cellularnuclei, such as nucleic acids, proteins or cytotoxic agents, but also ofother (therapeutically useful) compounds, are of considerable interestin the field of biotechnology. These techniques may be suitable fortransport and translation of nucleic acids into cells in vitro and invivo and thus for protein or peptide production, for regulation of geneexpression, for induction of cytotoxic or apoptotic effects, foranalysis of intracellular processes and for the analysis of the effectof the transport of a variety of different cargos into a cell (or cellnucleus), etc.

One important application of such a transfer of a cargo of interest fromthe external medium into tissue or cells is gene therapy, wherein thecargo is typically a nucleic acid or a gene. Although this technique hasshown some rather promising developments in the last decades, genetransfer is typically limited by the inability of the gene transfervectors to effectively transfer the biologically active cargo into thecytoplasm or nuclei of cells in the host to be treated without affectingthe host genome or altering the biological properties of the activecargo.

In this respect, several techniques have been developed in an effort tomore efficiently transfect e.g. nucleic acids, such as DNA or RNA, intocells. Transfection of nucleic acids into cells or tissues of patientsby methods of gene transfer is a central method of molecular medicineand plays a critical role in therapy and prevention of numerousdiseases.

Representative examples of gene transfer methods include general(physical or physico-chemical) methods such as coprecipitating nucleicacids with calcium phosphate or DEAE-dextran, a method which enablesnucleic acids to penetrate the plasma membrane and then enter the celland/or nucleus. However, this technique suffers from low transferefficiency and a high percentage of cell death. Additionally, thismethod is restricted to in vitro or ex vivo methods, but is notapplicable to in vivo situations due to its very nature.

The same holds for methods involving in vitro electroporation. In vitroelectroporation is based on the use of high-voltage current to make cellmembranes permeable to allow the introduction of new nucleic acids, e.g.DNA or RNA, into the cell. However, such methods are typically notsuitable in vivo. Furthermore, this technique also suffers from lowtransfer efficiency and a high percentage of cell death.

Further well known physical or physico-chemical methods include (direct)injection of (naked) nucleic acids or biolistic gene transfer. Biolisticgene transfer (also known as biolistic particle bombardment) is a methoddeveloped at Cornell University that allows introducing genetic materialinto tissues or culture cells. Biolistic gene transfer is typicallyaccomplished by surface coating metal particles, such as gold or silverparticles, and shooting these metal particles, comprising the adsorbedDNA, into cells by using a gene gun. Similar as discussed above thismethod is restricted to in vitro or ex vivo methods, but is usually notapplicable in in vivo situations.

Other methods utilize the transport capabilities of so calledtransporter molecules. Transporter molecules to be used in this contexttypically may be divided into viral vectors, i.e. transporter molecules,which involve viral elements, and nonviral vectors.

The most successful gene therapy strategies available today rely onviral vectors, such as adenoviruses, adeno-associated viruses,retroviruses, and herpes viruses. These viral vectors typically employ aconjugate of a virus-related substance with a strong affinity for DNAand a nucleic acid. Due to their infection properties, viruses or viralvectors have a very high transfection rate. The viral vectors typicallyused are genetically modified in a way that no functional infectiousparticles are formed in the transfected cell. In spite of this safetyprecaution, however, there are many problems associated with viralvectors related to immunogenicity, cytotoxicity, and insertionalmutagenesis. As an example, the risk of uncontrolled propagation of theintroduced therapeutically active genes or viral genes cannot be ruledout, e.g., because of possible recombination events. Additionally, theviral conjugates are difficult to use and typically require a longpreparation prior to treatment (see, e.g., U.S. Pat. No. 5,521,291).

Although nonviral vectors are not as efficient as viral vectors, manyhave been developed to provide a safer alternative in gene therapy. Someof the most common nonviral vectors include polyethylenimine,dendrimers, chitosan, polylysine, and peptide based transporter systems,e.g. many types of peptides, which are generally cationic in nature andable to interact with nucleic acids such as plasmid DNA throughelectrostatic interactions.

For successful delivery, the nonviral vectors, particularly peptidebased transporter systems must be able to overcome many barriers. Suchbarriers include protection of the cargo moiety, e.g. of DNA or othercompounds, during transport and prevention of an early degradation ormetabolisation of the cargo moiety in vivo. In case of nucleic acids,such as DNA and RNA molecules, the nonviral vectors must furthermore becapable to specifically deliver these molecules for efficient geneexpression in target cells.

Particularly for nucleic acids such DNA and RNA molecules there arepresently 4 barriers nonviral vectors must overcome to achievesuccessful gene delivery (see e.g. Martin et al., The AAPS Journal 2007;9 (1) Article 3). The nonviral vector must be able to 1) tightly compactand protect the nucleic acids, 2) it must able to target specificcell-surface receptors, 3) the nonviral vector must be capable todisrupt the endosomal membrane, and 4) it has to deliver the nucleicacid cargo to the nucleus and allow translation of an encoded protein orpeptide sequence.

Such nonviral vectors, particularly peptide-based nonviral vectors, areadvantageous over other nonviral strategies in that they are in generalable to achieve all 4 of these goals, however, with different efficiencyregarding the different barriers.

As an example, cationic peptides rich in basic residues such as lysineand/or arginine are able to efficiently condense nucleic acids such asDNA into small, compact particles that can be stabilized in serum.Furthermore, attachment of a peptide ligand to the polyplex allowstargeting to specific receptors and/or specific cell types. Polyplexesor cationic polymers as mentioned above typically form a complex withnegatively charged nucleic acids leading to a condensation of nucleicacids and protecting these nucleic acids against degradation. Transportinto cells using polyplexes (cationic polymers) typically occurs viareceptor mediated endocytosis. Thereby, the DNA is coupled to a distinctmolecule, such as Transferrin, via e.g. the polyplex poly-L-lysine(PLL), which binds to a surface receptor and triggers endocytosis.Polyplexes (cationic polymers) include e.g. poly-L-lysine (PLL),chitosan, polyethylenimine (PEI), polydimethylaminoethylmethacrylate(PD-MAEMA), polyamidoamine (PAMAM). Such effects are also known fromnanoplexes (nanoparticular systems) or lipoplexes (liposomal systems).Nanoplexes (nanoparticular systems) typically involve the use ofpolyacrylates, polyamides, polystyrene, cyanoacrylates, polylactat(PLA), poly(lactic-co-glycolic acid) (PLGA), etc. Lipoplexes orliposomal systems typically involve the use of cationic lipids, whichare capable to mimick a cell membrane. Thereby, the positively chargedmoiety of the lipid interacts with the negatively charged moiety of thenucleic acid and thus enables fusion with the cell membrane. Lipoplexesor liposomal systems include, e.g. DOTMA, DOPE, DOSPA, DOTAP, DC-Chol,EDMPC, etc.

In this context, receptor-mediated endocytosis is also widely exploitedin experimental systems for the targeted delivery of cargos such asnucleic acids or therapeutic agents into cells. During receptor-mediatedendocytosis the cargo-containing complexes are either selectivelyinternalized by receptors located in the cell membrane which arespecific for the cargos, or by specific antibodies located in membraneconstituents. Endocytotic activity has been described for many receptorsincluding IgG Fc, somatostatin, insulin, IGF-I and -II, transferrin,EGF, GLP-1, VLDL or integrin receptors, etc.

Different peptide or protein sequences have been tested widely for theiruse in gene transfer methods via receptor-mediated endocytosis.Interestingly, the isolation of peptide sequences that direct efficientreceptor-mediated endocytosis have been profoundly boosted by the use ofphage display technologies. Phage display libraries are extremelypowerful tools that provide for a practically unlimited source ofmolecular variants including modifications of natural ligands or cargomoieties to cell receptors and short peptides. Similar libraries havealso been injected directly into mice and peptide sequences have beensuccessfully isolated that show a 13-fold selectivity for brain andkidney.

Proprotein convertases may serve as an example of peptide or proteinsequences that may be used for transport of molecules into cells.Proprotein convertases are an example of a cell surface receptor whichgets internalized through receptor mediated endocytosis. These proteinshave been shown to be responsible for conversion of precursors ofpeptide hormones, neuropeptides, and many other proteins into theirbiologically active forms. All cleavage sites for the proproteinconvertase family obey to the consensus R-X-X-R. The mammalianproproteinconvertases can be classified into three groups on the basisof their tissue distribution. Furin, PACE4, PC5/PC6, andLPCIPC7/PC8/SPC7 are expressed in a broad range of tissues and celllines. In contrast, expression of PC2 and PC1/PC3 is limited toneuroendocrine tissues, such as pancreatic islets, pituitary, adrenalmedulla and many brain areas. Expression of PC4 is highly restricted totesticular spermatogenic cells. The neuroendocrine-specific convertases,PC2 and PC1/PC3, are mainly localized in secretory granules. PC5/PC6Ahas also been reported to be localized to secretory granules.Furthermore, indirect evidence has suggested that a proportion ofproprotein convertases molecules is present on the cell surface, and ithas been shown that furincycles between the TGN and the cell surface.Taken together, these properties indicate that proprotein convertasestransport extracellular ligands into the intracellular space.

Advantageous are also so called translocatory proteins or of proteintransduction domains (PTDs). Peptide sequences derived fromtranslocatory proteins or protein transduction domains (PTDs) aretypically able to selectively lyse the endosomal membrane in its acidicenvironment leading to cytoplasmic release of the polyplex.Translocatory proteins are considered as a group of peptides capable ofeffecting transport of macromolecules between cells (translocatoryproteins), such as HIV-1 TAT (HIV), antennapedia (Drosophilaantennapedia), HSV VP22 (Herpes simplex), FGF or lactoferrin, etc. Incontrast, protein transduction domains (PTDs) are considered as a groupof peptides capable of directing proteins and peptides covalently boundto these sequences into a cell via the cell membrane (Leifert andWhitton: Translocatory proteins and protein transduction domains: acritical analysis of their biological effects and the underlyingmechanisms. Molecular Therapy Vol. 8 No. 1 2003). Common totranslocatory proteins as well as to PTDs is a basic region, which isregarded as mainly responsible for transport of the fusion peptidessince it is capable of binding polyanions such as nucleic acids. Withoutbeing bound thereto, PTDs may act similar to cationic transfectionreagents using receptor dependent non-saturatable adsorptiveendocytosis. PTDs are typically coupled to proteins or peptides in orderto effect or enhance a CTL response when administering a peptide basedvaccine (see review: Melikov and Chernomordik, Arginine-rich cellpenetrating peptides: from endosomal uptake to nuclear delivery, Cell.Mol. Life Sci. 2005).

Unfortunately, peptide based transporter systems typically undergoproteolytic degradation in vivo due to peptidases leading to truncatedtransporter (and/or cargo) sequences. Such peptidases may bedistinguished into exopeptidases and endopeptidases, which are bothenzymes capable of catalysing the splitting of proteins into smallerpeptide fractions and even into single amino acids by a process known asproteolysis. In this context, endopeptidases are typically proteolyticpeptidases that break peptide bonds of nonterminal amino acids (i.e.within the molecule). Endopeptidases are typically specific for certainamino acids. Examples of endopeptidases include e.g. trypsin,chymotrypsin, elastase, thermolysis, pepsin and endopeptidase V, etc.Trypsin is known to cut after Arg or Lys, unless followed by a Pro.Chymotrypsinis known to cut after Phe, Trp, or Tyr, unless followed by aPro. Chymotrypsincuts more slowly after Asn, His, Met or Leu. Elastasecuts after Ala, Gly, Ser, or Val, unless followed by a Pro. Thermolysinis a heat stable endoprotease, which cuts before Ile, Met, Phe, Trp,Tyr, or Val, unless preceded by Pro. Thermolysin sometimes cuts afterAla, Asp, His or Thr. Pepsin is known to cut before Leu, Phe, Trp orTyr, unless preceded by Pro. Finally, endopeptidase V8 is known to cutafter Glu. In contrast to endopeptidases exopeptidases are enzymes thatcatalyse the removal of an amino acid from the end of a polypeptidechain and thus cleave the end of said polypeptide chain. Exopeptidasesmay be distinguished from their cleavage site into aminopeptidases andcarboxypeptidases. Aminopeptidases are typically zinc-dependent enzymesand are produced by glands of the small intestine Aminopeptidasesusually cleave a single amino acid from the amino-terminal end of apeptide or protein sequence. Carboxypeptidases are typically enzymesthat hydrolyze the carboxy-terminal (C-terminal) end of a peptide bond.Humans, animals, and plants contain several types of carboxypeptidaseswith diverse functions ranging from catabolism to protein maturation,which is a digestive enzyme present in pancreatic juice, will cleave asingle amino acid from the carboxylic end of the peptide. A particularexample is Carboxypeptidase N (CPN), a plasma zinc metalloproteasecomprised of two small subunits that have enzymatic activity, and twolarge subunits, which protect the enzyme from degradation. CPN cleavesthe carboxyl-terminal amino acids arginine and lysine from biologicallyactive peptides such as complement anaphylatoxins, kinins, andfibrinopeptides.

In order to modify proteolytic cleavage by peptide based transportersystems as defined above, the peptide based transporter systems may becomposed entirely of D-amino acids, thereby forming “retro-inversopeptide sequences”. The term “retro-inverso (peptide) sequences” refersto an isomer of a linear peptide sequence in which the direction of thesequence is reversed and the chirality of each amino acid residue isinverted (see e.g. Jameson et al., Nature, 368, 744-746 (1994); Brady etal., Nature, 368, 692-693 (1994)). The advantage of combiningD-enantiomeric amino acids and reverse synthesis is that the positionsof carbonyl and amino groups in each amide bond are exchanged, while theposition of the side-chaingroups at each alpha carbon is preserved. Dueto the conformational change of the naturally occurring L-enantiomericamino acids of the peptide sequence of a peptide based transporter toD-enantiomeric amino acids the risk of proteolytic cleavage in vivo iseliminated, being advantageous and highly efficient for the purpose oftransfection of a cargo moiety into a cell. In contrast, the term“reverse sequence” refers to a sequence in which the direction of thesequence is reversed (but the chirality of each amino acid residue isnot inverted (e.g. D-Arg-L-Arg-L-Arg→L-Arg-L-Arg-D-Arg).

However, though efficiently working as a transporter molecule as definedabove the conformational change of the naturally occurringL-enantiomeric amino acids of the peptide sequence of such a peptidebased transporter to D-enantiomeric amino acids entails the risk of apredominant accumulation of these transporters in the cell during thewhole lifetime of a cell or even longer in the (surrounding) tissue ororganism. Accordingly, such transporters, even if the attached cargomoiety is cleaved off or is metabolised in the meantime, may remain inthe cell and participate in further inter- and intracellular processesleading to unknown and unwanted side effects.

SUMMARY

Accordingly, there is a need in the art to provide alternative nonviraltransporter molecules, preferably peptide based transporter systems asdefined above, which avoid such an unwanted accumulation in the cell ortissue but nevertheless allow an efficient transfer of cargo moietiesinto cells.

The above object is solved by the subject matter as defined in theclaims attached hereto, particularly by a novel transporter constructand its conjugates (transporter cargo conjugate molecule) as defined inthe claims. The above object is furthermore solved by methods and usesemploying the novel transporter construct and its conjugates as definedin the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figures are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

FIG. 1 depicts the results of a quantitative degradation assay withProteinase K involving several transporter constructs with poly-Argsequences, each showing a different pattern of D- and L-amino acids(D-/L-pattern). The transporter constructs used in this assay weretermed 1- to 6-. The different D-/L-pattern of the sequences isdescribed using capitals and minor letters. The capitals in thesesequences (“R”-amino acids) refer to L-enatiomeric arginine (L-Arg) andthe minor letters in these sequences (“r”-amino acids) refer toD-enatiomeric arginine (L-Arg). The sensitivity to Proteinase K wasmeasured at time intervals t=0, 10 and 40 minutes. The results of thequantitative degradation assay with Proteinase K were then determined onthe basis of samples taken at these specific time intervals. Thesesamples were analyzed utilizing a mass spectrometry analysis usingstarting material ionintensities as reference values. As a conclusion,when a stretch of 3 or more L-Arg is present in the sequence the peptideshows sensitivity to Proteinase K and is degraded. Peptides with 2 orless L-Arg next to each other are not degraded and results arecomparable to full-D sequence or D-TAT (SEQ ID NO: 251), respectivelyD-JNKi.

FIG. 2: shows in a table a comparison of four different transporterconstructs of D-/L-TAT derivatives (termed L-TAT (SEQ ID NO: 18), r3-TAT(also termed r3-L-Tat; SEQ ID NO: 20), r3-TATi (also termed r3-L-TATi:SEQ ID NO: 21), and D-TAT; SEQ ID NO: 251), each having a length of 9amino acids but a different D-/L-pattern. The different D-/L-pattern ofthe sequences is described using capitals and minor letters. Thecapitals in the sequences (“R”-amino acids) refer to L-enatiomericarginine (L-Arg) and the minor letters in these sequences (“r”-aminoacids) refer to D-enatiomeric arginine (L-Arg). The table shown in FIG.2 illustrates the amino acid sequences of these TAT derived transporterconstructs with respect to their molecular weight (Mw) and pI-values.

FIG. 3 shows the results of a digestion of TAT derived transporterconstructs in 10% and 50% human serum at 37° C. until completedegradation of these TAT derived transporters. The TAT-derivedtransporter constructs are as described in FIG. 2, wherein thetransporter constructs r3-TAT (also termed r3-L-Tat; SEQ ID NO: 20),r3-TATi (also termed r3-L-TATi; SEQ ID NO: 21) were additionallyprotected N-terminally with a beta-Alanine. As can be seen in FIG. 3,D-TAT transporter constructs are protease resistant, whereas L-TATtransporter constructs are degraded too early in vivo in order to effectan efficient transport into cells. Only transporter constructs r3-TAT(also termed r3-L-Tat; SEQ ID NO: 20), r3-TATi (also termed r3-L-TATi,SEQ ID NO: 21) show a degradation within a suitable time limit, allowingto limit the in vivo stability for therapeutic applications.

FIG. 4 shows the results of a digestion of TAT derived transporterconstructs in 10% and 50% human serum at 37° C. until completedegradation of these TAT derived transporters under involvement ofbeta-Alanine (b-Ala) in protecting the peptides. Therefore, beta-Ala wasadded to the N-terminus of TAT derived transporter constructs L-TAT asL-TATi as already described in FIG. 3. As can be seen in FIG. 4N-terminal protection of the transporter peptides with beta-Alanine doesnot lead to a significant effect, i.e. an improved stability.

FIG. 5 depicts the results of the time dependant internalization(uptake) of FITC-labeled TAT derived transporter constructs into cellsof the HL-60 cell line. HL-60 cells were incubated 30 min, 1, 6 or 24hours with 101.1M of the TAT-derivative transporters. The cells werethen washed twice with an acidic buffer (0.2 M Glycin, 0.15 M NaCl, pH3.0) and twice with PBS. Cells were broken by the addition of RIPA lysisbuffer. The relative amount of internalized peptide was then determinedby reading the fluorescence intensity (Fusion Alpha plate reader;PerkinElmer) of each extract followed by background substraction andprotein content normalization. The r3-L-TAT transporter construct (SEQID NO: 20) showed an internalization capability as effective as theD-TAT transporter construct. The r3-L-TATi transporter construct (SEQ IDNO: 21), which internalized in a time dependent manner, as both previoustransporters, seems to be less efficient but still suitable, whereasL-TAT (SEQ ID NO: 18) doesn't accumulate over a period of 24 hours.

FIG. 6 shows results of a confocal microscopy of of cells treated withfluorescently labeled TAT derivative transporters. The dissociatedcortical primary neurons from P2 Sprague Dawley rats were cultured 12days in neurobasal medium before exposure 24 hours to 500 nM of theFITC-labeled TAT derivative transporters. The cells were washed fivetimes with PBS on ice and then mounted in fluorsave mounting mediumwithout prior fixation. Acquisitions were performed on LSM510metaconfocal microscope (Zeiss). Images were processed with LSM510software and mounted using Adobe photoshop. Visualization by confocalmicroscopy of labeling with 500 nM FITC-transporters (A: green). Nucleiwere stained by Hoechst (B: blue). The r3-L-TAT (SEQ ID NO: 20) as wellas the D-TAT (SEQ ID NO: 251) and the r3-L-TATi (SEQ ID NO: 21)transporter constructs were internalized into the cytoplasm of the nonstressed neurons (C: Merge panel). However, after 24 hours incubation,the L-TAT transporter (SEQ ID NO: 18) was not present anymore.

FIG. 7 illustrates the uptake (internalization) of FITC-labeled TATderived transporter constructs in vitro (10 μM, HepG2 hepatocarcinoma,HCT-116 tumoral colon, 24 h). The constructs used were different TATderived transporter constructs termed D-TAT (SEQ ID NO: 251) and r3-TATi(also termed r3-L-TATi, SEQ ID NO: 21), each having a length of 9 aminoacids but a different D-/L-pattern, and the test constructs r₆R₃ (SEQ IDNO: 260) and DAK, wherein the constructs additionally have been labeledwith beta-Alanine at their N-terminus. As can be seen, uptake was mostefficient for constructs D-TAT (SEQ ID NO: 2.50 and r₆R₃ (SEQ ID NO:260), followed by r3-L-TATi (SEQ ID NO: 21).

FIG. 8 shows the uptake (internalization) of FITC-labeled TAT derivedtransporter constructs in vitro (10 μM, U937, Lymphoma, 24 h). Theconstructs used were four different TAT derived transporter constructs(termed L-TAT, SEQ ID NO: 18), r3-TAT (also termed r3-L-Tat, SEQ ID NO:20), r3-TATi (also termed r3-L-TATi, SEQ ID NO: 21), and D-TAT, SEQ IDNO: 251), each having a length of 9 amino acids but a differentD-/L-pattern. Additionally, the construct DAK was used for comparisonand a control sample, containing only the amino acids D, A and K. As canbe seen, the uptake of r3-TAT (SEQ ID NO: 201, r3-TATi (SEQ ID NO: 21)and D-TAT (SEQ ID NO: 251) transporter constructs into the cells wasmost efficient, wherein L-TAT (SEQ ID NO:18) showed a significantlylower uptake into the cells.

FIG. 9 shows that the uptake (internalization) of the D-TAT transporterconstruct is HSPG-dependent at a concentration of 500 nM over 24 hoursin U937 cells, Lymphoma. The construct used for the experiment was D-TAT(SEQ ID NO: 251), having a length of 9 amino acids and being labeledwith FITC and at its N-terminus with beta-Alanine.

FIG. 10 shows that an exit of the FITC-labeled TAT derived transporterconstructs is not observed in U937 cells at 500 nM FITC-D-TAT. Theconstruct used for the experiment was D-TAT (SEQ ID NO: 251), having alength of 9 amino acids and being labeled with FITC.

FIG. 11 shows that an exit of the FITC-labeled TAT derived transporterconstructs is observed at 10 μM FITC-D-TAT, and is HSPG-dependent (U937,lymphoma). The construct used for the experiment was D-TAT (SEQ ID NO:251), having a length of 9 amino acids and being labeled with FITC andat its N-terminus with beta-Alanine.

FIG. 12 shows that an uptake (internalization) and an exit of theFITC-labeled TAT derived transporter constructs is observed at 10 μMFITC-D-TAT in non WBC-lines (white blood cells lines). The constructused for the experiment was D-TAT (SEQ ID NO: 251), having a length of 9amino acids and being labeled with FITC.

FIG. 13A and FIG. 13B show internalizations experiments using TATderived transporter constructs of general formula (I). FIG. 13A showspeptide numbers 1-48 and FIG. 13B shows peptide numbers 49-96. As can beseen in FIGS. 13A and 13B, after 24 hours incubation, all transporterswith the consensus sequence rXXXrXXXr (SEQ ID NO: 252; see above for aselection of potential sequences) showed a higher internalizationcapability than the L-TAT transporter (SEQ ID NO: 18). Hela cells wereincubated 24 hours in 96 well plate with 10 mM of the r3-L-TAT-derivedtransporters. The cells were then washed twice with an acidic buffer(0.2M Glycin, 0.1.5M NaCl, pH 3.0) and twice with PBS. Cells were brokenby the addition of RIPA lysis buffer. The relative amount ofinternalized peptide was then determined by reading the fluorescenceintensity (Fusion Alpha plate reader; PerkinElmer) of each extractfollowed by background subtraction.

FIG. 14A-D show internalizations experiments using TAT derivedtransporter constructs of general formula (I). As can be seen in FIG.14A-D, one position appears to be critical for highest transporteractivity and for improved kinetics of transport activity: Y in position2 (peptide No 91 corresponding to SEQ ID NO: 116). Briefly, Hela cellswere incubated 2, 6 or 24 hours in 24 well plate with increasing dose ofthe r3-L-TAT-derivative transporters (0, 500 nM, 1 mM or 10 mM). Thecells were then washed twice with an acidic buffer (0.2M Glycin, 0.15MNaCl, pH 3.0) and twice with PBS. Cells were broken by the addition ofRIPA lysis buffer. The relative amount of internalized peptide was thendetermined by reading the fluorescence intensity (Fusion Alpha platereader; PerkinElmer) of each extract followed by background substation.

FIG. 15A and FIG. 15B show Fluorescent TAT derivative transporters D-TAT(SEQ ID NO: 251)-FITC or r3-L-TAT (SEQ ID NO: 20)-FITC target differenthuman leukocyte populations, respectively. FIG. 15A shows D-TAT (SEQ IDNO: 251)-FITC. The percentage of cells gated in the respective quadrantsis as follows (given clockwise beginning with upper left quadrant):

Monocytes (CD14) 9,24; 19,37; 31,71; 39,68; Neutrophils (CD15) 17,03;13,87: 21,53; 47,57; Lymphocytes T (CD3) 22,7; 11,82; 18,01; 47,46;Lymphocytes B (CD19) 32,40; 2,12; 8,26; 57,22.

FIG. 15B shows r3-L-TAT (SEQ ID NO: 20)-FITC. The percentage of cellsgated in the respective quadrants is as follows (given clockwisebeginning with upper left quadrant):

Monocytes (CD14) 6,34; 16,65; 36,24; 40,77; Neutrophils (CD15) 11,74;13,75; 24,76; 49,75; Lymphocytes T (CD3) 20,64; 8,96; 20,04; 50,36;Lymphocytes B (CD19) 27,83; 1,76; 8,48; 61,92.

FIG. 16 table indicates the mean fluorescence values for fluorescent TATderivative transporters D-TAT (SEQ ID NO: 251)-FITC or r3-L-TAT (SEQ IDNO: 20)-FITC in each cell type as shown in FIG. 15 (FITC channel).

FIG. 17 shows uptake of selected transporter constructs according to thepresent invention by different cell types. Uptake is normalized versusD-TAT (SEQ ID NO: 251). HepG2: Hepatocarcinoma cells (human; nonleucocyte cell line); A549: Lung epithelial cells (human; non leucocytecell line); Raw: Macrophage cells (mouse; leucocyte cell line); J77:Macrophage cells (mouse; leucocyte cell line); BMDM: Bone Marrow-DerivedMacrophages (mouse, purified primary lencocytes). *n=2 independentexperiments (in duplicate) (except fOr peptide #64 n=1 in duplicate);**n=2 experiment (in duplieate) (except for peptide #64 n=2 induplicate); ***n=1 experiment (in duplicate).

FIG. 18 shows uptake of selected transporter constructs according to thepresent invention by different cell types. Uptake is normalized versusr₃-L-TAT (SEQ ID NO: 20). HepG2: liepatocarcinoma cells (human; nonleucocyte cell line); A549: Lung epithelial cells (human; non leucocytecell line); Raw: Macrophage cells (mouse; leucocyte cell line); J77:Macrophage cells (mouse; leucocyte cell line); BMDM: Bone Marrow-DerivedMacrophages (mouse; purified primary leucocytes). *n=2 independentexperiments (in duplicate) (except or peptide #64 n=1 in duplicate);**n=2 experiment (in duplicate) (except for peptide #64 n=2 induplicate); ***n=1 experiment (in duplicate).

DETAILED DESCRIPTION

According to a first aspect of the present invention, the object of thepresent invention is solved by a novel transporter construct comprisingor consisting of at least one sequence of the generic formula (I) (SEQID NO: 1):

D_(l)LLL_(x)D_(m)(LLL_(y)D_(n))_(a)

-   wherein: D is a D-amino acid;    -   L is a L-amino acid;    -   a is 0-3, preferably 0-2, more preferably 0, 1, 2 or 3, even        more preferably 0, 1, or 2 and most preferably 1;    -   l, m and n are independently from each other 1 or 2, preferably        1;    -   x and y are independently from each other 0, 1 or 2, preferably        1.

As used herein, the term “transporter construct” refers to an amino acidcontaining compound, which is capable of translocation across biologicalmembranes. As used herein, the term “trafficknig sequence” (or“transporter sequence”) refers to a sequence of amino acids providingtranslocation across biological membranes. Accordingly, the transporterconstructs according to the present invention comprise a traffickingsequence which allow the transporter construct to translocate acrossbiological membranes. Thus, the novel transporter constructs accordingto generic formula (I) effectively allow and may provide fOr thetransport of cargo moieties, e.g. of proteins or peptides, of nucleicacids, of small organic molecules, of antigens, of cytotoxic agents,etc., into an organism, a tissue, a cell (e.g. to be treated), acellular subcompartiment and/or into the nucleus of a cell.

Advantageously, the inventive transporter construct according to genericformula (I) is stable enough to prevent degradation by proteases priorto transport of the cargo moiety to its target site. On the other handside, the inventive transporter constructs according to generic formula(I) are not permanently persistent in the cell and may be degraded byproteases within a considerable time limit so as to avoid negative sideeffects such as unwanted accumulation of the novel inventive transporteror its conjugate in the cell. As surprisingly found by the inventors,such advantageous properties may be conferred to a trafficking sequence,particularly to any trafficking sequence known in the art only by theinventive pattern (herein also described as D-/L-pattern) of the abovedefined generic formula (I), the specific content and position of theD-amino acids in alteration with the specific content of L-amino acidsas defined ingeneric formula (I). This inventive D-/L-pattern allows askilled person to define the in vivo or in vitro persistence of theinventive novel transporter construct as defined above in the cellprecisely enough as a time sufficiently long to ensure administrationand entering of the inventive novel transporter construct into the cellor nucleus prior to degradation of the inventive novel transporterconstruct by proteases within a considerable time limit. This in vivo orin vitro persistence of the inventive novel transporter construct in thecell is in fact dependent on the specific content and position of theD-amino acids inalteration with the specific content of L-amino acids asdefined in generic formula (I). Furthermore, a transporter constructexhibiting the inventive D-/L-pattern of the above defined genericformula (I) is short enough to avoid a sterical hindrance of a cargomoiety by the inventive novel transporter construct in a transportercargo conjugate molecule such as defined below. It also allows a costefficient preparation of such inventive novel transporter constructs.Additionally, a conjugate of the transporter peptide or proteinof theabove defined generic formula (I) with either proteins or peptides,nucleic acids such as DNA and RNA molecules or with cytotoxic agents oreven small organic molecules, etc., may be formed easily.

According to the above defined generic formula (I), the inventive noveltransporter construct comprises L-amino acids and D-amino acidsaccording to the specific D-/L-pattern as set forth in generic formula(I).

In the context of the present invention L-amino acids, also termedL-enantiomeric amino acids, are preferably amino acids selected fromnatively occurring amino acids or their derivatives. Naturally occurringamino acids are typically selected from the standard (proteinogenic)amino acids alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutaminic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenyl alanine, proline, serine, threonine,tryptophane, tyrosine, and valine, as well as from non-standard aminoacids such as ornithine, citrulline, homocysteine, S-adenosylmethionione, hydroxyproline, selenocysteine, pyrrolysine, lanthionine,2-aminoisobutyric acid, dehydroalanine, gamma-aminobutyric acid, etc.

Derivatives from such L-amino acids or L-enantiomeric amino acidstypically comprise any naturally or non-naturally occurring derivativeof these amino acids, including, without being limited thereto, aminoacids as defined above comprising post-translational modifications orsynthetic modifications, including acetylation (at the N-terminus of thepeptide sequence, at lysine residues, etc.), deacetylation, alkylation,such as methylation, ethylation, etc. (preferably at lysine or arginineresidues within the peptide sequence), dealkylation, such asdemethylation, deethylation, etc., amidation (preferably at theC-terminus of the peptide sequence), formylation, gamma-carboxylation,glutamylation, glycosylation (preferably at asparagine, lysine,hydroxylysine, serine or threonine residues, etc., within the peptidesequence), addition of a heme or haem moiety, hydroxylation, iodination,isoprenylation addition of an isoprenoid moiety such as farnesyl orgeranylgeraniol, etc.), lipoylation (attachment of lipoatefunctionality), such as prenylation, formation of a GPI anchor,including myristoylation, farnesylation, geranylgernaylation, etc.,oxidation, phosphorylation (e.g. to a serine, tytosine, threonine or ahistidine moiety, etc., within the peptide sequence), sulfation (e.g. oftyrosine), selenoylation, sulfation, etc.

Derivatives of L-amino acids also include, without being limitedthereto, modified L-amino acids, which have been modified by introducingone of the following labels:

-   -   (i) radioactive labels, i.e. radioactive phosphorylation or a        radioactive label with sulphur, hydrogen, carbon, nitrogen,        etc.;    -   (ii) colored dyes (e.g. digoxygenin, etc.);    -   (iii) fluorescent groups (e.g. fluorescein, rhodamine,        flourochrome proteins as defined below, etc.);    -   (iv) chemoluminescent groups;    -   (v) a combination of labels of two or more of the labels        mentioned under (i) to (iv).

Particularly specific examples of derivatives of L-amino acids include,without being limited thereto, AMC (aminomethylcoumarin), Dabcyl(dimethylaminophenylazobenzoyl), Dansyl(dimethylaminonaphtalenesulfonyl), FAM (carboxyfluoroscein), Mca(methoxycoumarin acetyl), Xan (xanthyl), Abu (aminobutyric acid),Beta-Ala (beta-alanine), E-Ahx (6-aminohexanoic acid), Alpha-Aib(alpha-aminoisobutyric acid), Ams (aminoserine), Cha (cyclohexylamine),Dab (diaminobutyric acid), Hse (homoserine), Hyp (hydroxyproline), Mpr(mercaptopropionic acid), Nal (naphtylalanine), Nva (Norvaline), Orn(ornithine), Phg (phenylglycine), Sar (sarcosine), Sec (selenocysteine),Thi (thienylalanine), etc.

Furthermore, L-enantiomeric amino acids selected for the inventive noveltransporter construct as defined above furthermore may be selected fromspecific combinations of the above defined L-enantiomeric amino acids orderivatives thereof. Such combinations may comprise 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 or even more of the above defined L-enantiomeric amino acidsor derivatives thereof. Combinations are also possible between any ofthe above defined L-enantiomeric amino acids or derivatives thereof andany the above defined D-enantiomeric amino acids or derivatives thereofwithin the definitions of generic formula (I) or of any of subformulasas defined herein. Such specific combinations of amino acids may exhibita higher or a lower stability towards peptidases and thus may provide afurther possibility to render the in vivo or in vitro stability of theinventive novel transporter construct as defined above towards a higheror a lower stability. As an example, the inventive novel transporterconstruct may contain the dipeptide sequence Arg-Lys inD- and/or L-form(i.e. both as D-enantiomeric amino acids or as L-enantiomeric aminoacids or mixed D- and L-enantiomeric amino acids), preferably in L-form,wich exhibits a lower stability towards pepidases and thus may be usedto destabilize the peptide sequence of the inventive novel transporterconstruct and therefore to decrease its half life in vivo to a furtherextent.

In the context of the present invention D-amino acids, also termedD-enantiomeric amino acids, are preferably non-native(non-proteinogenic) “retro-inverso” amino acids, wherein thesenon-native (non-proteinogenic) “retro-inverso” amino acids arepreferably derived from naturally occurring L-amino acids and/or theirderivatives as defined above. In this context, the term “retro-inverso”refers to an isomer of a naturally occurring L-amino acid as definedabove (and peptides made therefrom) in which the chirality of thenaturally occurring L-amino acid residue is inverted in thecorresponding D-amino acid (see e.g. Jameson et al., Nature, 368,744-746 (1994); Brady et al., Nature, 368, 692-693 (1994)). In otherwords, in the peptide bonds of D-amino acids the positions of carbonyland amino groups are exchanged, while the position of theside-chaingroups at each alpha carbon is preserved. Accordingly, D-aminoacids may be inserted into a peptide sequence consisting of orcomprising L-amino acids and therefore may be conjugated with L-aminoacids as defined above by methods known in the art. Such methods knownin the art include e.g., without being limited thereto, liquid phasepeptide synthesis methods or solid peptide synthesis methods, e.g. solidpeptide synthesis methods according to Merrifield, t-Boc solid-phasepeptide synthesis, Fmoc solid-phase peptide synthesis, BOP(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate) based solid-phase peptide synthesis, etc. Thecontent of D-amino acids in the inventive novel transporter constructsaccording to the D-/L-pattern of generic formula (I) above additionallyprovides a further variety of useful properties. For example, such noveltransporter constructs enter cells more efficiently and are more stable(especially in vivo) and show lower immunogenicity than correspondingL-amino-acid-sequence based transporter constructs. However, they arenot as persistent in the cell as transporter constructs entirely made ofD-amino acids, particularly due to the fact that almost alldecomposition enzymes, like proteases or peptidases, cleave peptidebonds between adjacent L-amino acids. Consequently, peptides composed ofD-enantiomeric amino acids and L-enantiomeric amino acids are largelyresistant towards a fast proteolytic breakdown without leading to anaccumulation in the cell due to a missing degradation by proteases.

The above defined inventive novel transporter construct according togeneric formula (I), preferably comprises L-amino acids and D-aminoacids or their derivatives as defined above. Such derivatives may becontained in the entire inventive novel transporter construct in acontent of about 0%, about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, or even about 100%. Inother words, the entire inventive novel transporter peptide may containabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, or even more, of such derivatives, whereinthe maximum number of possible derivatives is, of course, limited by themaximum number of amino acids as contained in the above definedinventive novel transporter construct according to generic formula (I).

According to the above defined generic formula (I), the inventive noveltransporter construct comprises a specific D-/L-pattern of L-amino acidsand D-amino acids, which is defined by integers a, l, m, n, x and y.

According to the above definition of generic formula (I), a is adeterminant defining the number of repetitions of the subgroup(LLL_(y)D) as defined in the generic formula (I)D_(l)LLL_(x)D_(m)(LLL_(y)D_(n))_(a). According to the definition above,a may be any number selected from the range 0-3, preferably selectedfrom the range 0-2, more preferably selected from the range 0-1, or maybe selected from individual numbers 0, 1, 2 or 3, even more preferablyfrom individual numbers 0, 1, or 2 and most preferably a=1. According tothe specific number of repetitions of a=0, 1, 2 or 3, the inventivenovel transporter construct according to generic formula (I)D_(l)LLL_(x)D_(m)(LLL_(y)D_(n))_(a), may consist or comprise at leastone of the following subformulas (Ia) to (Id):

(Ia): (SEQ ID NO: 2) D_(l)LLL_(x)D_(m); (Ib): (SEQ ID NO: 3)D_(l)LLL_(x)D_(m)LLL_(y)D_(n); (Ic): (SEQ ID NO: 4)D_(l)LLL_(x)D_(m)LLL_(y)D_(n)LLL_(y)D_(n) or (Id): (SEQ ID NO: 5)D_(l)LLL_(x)D_(m)LLL_(y)D_(n)LLL_(y)D_(n)LLL_(y)D_(n).

Furthermore, according to the above definition of generic formula (I),l, m and n are integers defining the number of D-amino acids occurringin generic formula (I), but also in the subformulas (Ia) to (Id) asdefined herein. Integers l, m and n may be selected independently fromeach other. This particularly holds for determinant n, which may occurseveral times in generic formula (I) or subformulas (Ia) to (Id) asdefined herein, i.e. if n occurs several times, each n may be selectedindependently from each other. According to the definition above,integers 1, m and n independently of each other may be any numberselected from the range 1-2, or may be selected from individual numbers1 or 2, even more preferably 1, m and/or n=1.

Additionally, according to the above definition of generic formula (I),x and y are integers defining the number of L-amino acids occurring inthe generic formula (I), but also in the subformulas (Ia) to (Id) asdefined herein. Integers x and y may be selected independently from eachother. According to the definition above, integers x and y independentlyof each other may be any number selected from the range 0-2, preferablyselected from the range 0-1, or may be selected from individual numbers0, 1 or 2, even more preferably from individual numbers 0 or 1 and mostpreferably x and/or y=1.

According to one particularly preferred embodiment, the object of thepresent invention is solved by a novel transporter construct comprisingor consisting at least one sequence of the specific subformula (Ie):

(SEQ ID NO: 6) DLLLD(LLLD) _(a);wherein D, L, and a are as defined above for generic formula (I) orsubformulas (Ia) to (Id). According to another particularly preferredembodiment, the object of the present invention is solved by a noveltransporter construct comprising or consisting at least one sequence ofthe specific subformula (If):

(SEQ ID NO: 7) DLLLDLLLD;wherein D and L are as defined above for generic formula (I) orsubformulas (Ia) to (Id).

The inventive novel transporter construct according to generic formula(I) or according to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or(If), particularly transporter constructs comprising the novelD-/L-pattern may be used with or be applied to any trafficking sequenceknown in the art, wherein the selected number of contiguous amino acidsof those trafficking sequences is determined by the number of aminoacids as defined by generic formula (I) or any of subformulas (Ia),(Ib), (Ic), (Id), (Ie), or (If). Such trafficking sequences typicallydirect the transport of a cargo moiety into a cell or the nucleus or afurther specific target region and may comprise, without being limitedthereto, translocatory proteins as defined above, e.g. derived from HIVTAT (HIV), e.g. native proteins such as e.g. the TAT protein (e.g. asdescribed in U.S. Pat. Nos. 5,804,604 and 5,674,980, each of thesereferences being incorporated herein by reference), e.g. derived fromHIV tat (HIV), HSV VP22 (Herpes simplex) (described in e.g. WO 97/05265;Elliott and O'Hare, Cell 88: 223-233 (1997)), non-viral proteins(Jackson et al, Proc. Natl. Acad. Sci. USA 89: 10691-10695 (1992)),trafficking sequences derived from Antennapedia, particularly fromDrosophila antennapedia (e.g. the antennapedia carrier sequencethereof), FGF, lactoferrin, etc. or derived from basic peptides, e.g.peptides having a length of 5 to 15 amino acids, preferably 10 to 12amino acids and comprising at least 80%, more preferably 85% or even 90%basic amino acids, such as e.g. arginine, lysine and/or histidine, ormay be selected from e.g. arginine rich peptide sequences, such as R₉,R₈, R₇, R₆, R₅, etc., from VP22, from PTD-4 derived proteins orpeptides, from RGD-K₁₆, from PEPT1/2 or PEPT1/2 derived proteins orpeptides, from SynB3 or SynB3 derived proteins or peptides, from PCinhibitors, from P21 derived proteins or peptides, or from JNKI derivedproteins or peptides. Furthermore, variants, fragments and derivativesof one of the native proteins used as trafficking sequences aredisclosed herewith.

Particular examples of trafficking sequences forming a basis for thenovel transporter construct according to generic formula (I) or to anyof subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), as defined above,may be selected from, without being limited thereto, a so-called TATcell permeation sequence derived from the basic trafficking sequence ofthe HIV-1 TAT protein. Preferably, the basic trafficking sequence of theHIV-1 TAT protein may include sequences from the human immunodeficiencyvirus HIV-1 TAT protein, e.g. as described in, e.g., U.S. Pat. Nos.5,804,604 and 5,674,980, each incorporated herein by reference. In thiscontext, the full-length HIV-1 TAT protein has 86 amino acid residues

SEQ ID NO: 8

encoded by two exons of the HIV TAT gene. TAT amino acids 1-72 areencoded by exon 1, whereas amino acids 73-86 are encoded by exon 2. Thefull-length TAT protein is characterized by a basic region whichcontains two lysines and six arginines (amino acids 49-57) and acysteine-rich region which contains seven cysteine residues (amino acids22-37). The basic region (i.e., amino acids 49-57) was thought to beimportant for nuclear localization. Ruben, S. et al., J. Virol. 63: 1-8(1989); Hauber, J. et al., J. Virol. 63 1181-1187 (1989). Thecysteine-rich region mediates the formation of metal-linked dimers invitro (Frankel, A. D. et al, Science 240: 70-73 (1988); Frankel, A. D.et al., Proc. Natl. Acad. Sci USA 85: 6297-6300 (1988)) and is essentialfor its activity as a transactivator (Garcia, J. A. et al., EMBO J. 7:3143 (1988); Sadaie, M. R. et al., J. Virol. 63:1 (1989)). As in otherregulatory proteins, the N-terminal region may be involved in protectionagainst intracellular proteases (Bachmair, A. et al., Cell 56: 1019-1032(1989)). Preferred TAT trafficking sequences utilized with a to genericformula (I) or any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If),are preferably characterized by the presence of the TAT basic regionamino acid sequence (amino acids 49-57 of naturally-occurring tatprotein); the absence of the TAT cysteine-rich region amino acidsequence (amino acids 22-36 of naturally-occurring TAT protein) and theabsence of the TAT exon 2-encoded carboxy-terminal domain (amino acids73-86 of naturally-occurring TAT protein).

According to a more preferred embodiment the trafficking sequencesforming a basis for the novel transporter construct according to genericformula (I) or to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or(If), as defined above, may be selected from an amino acid sequencecontaining TAT residues 48-57 or 49 to 57, and most preferably a TATsequence according to any of SEQ ID NOs: 8 to 14, or from a generic TATsequence NH₂—X_(n) ^(b)-RKKRRQRRR-X_(n) ^(b)—COOH (L-generic-TAT (s))

SEQ ID NO: 16

and/or XXXXRKKRRQ RRRXXXX (L-generic-TAT)

SEQ ID NO: 15

. In this context, each X typically represents an amino acid residue,preferably selected from any (naturally occurring) amino acid residue asdefined herein. Furthermore, each X_(n) ^(b) may be selected from anyamino acid residue as defined herein, wherein n (the number ofrepetitions of X) is 0-5, 5-10, 10-15, 15-20, 20-30 or more. Preferably,X_(n) ^(b) represents a contiguous stretch of peptide residues derivedfrom the sequence according to SEQ ID NO: 8 (TAT (1-86)). Alternatively,the trafficking sequences forming a basis for the novel transporterconstruct according to generic formula (I) or to any of subformulas(Ia), (Ib), (Ic), (Id), (Ie), or (If), as defined above, may be selectedfrom, e.g., a peptide containing the amino acid sequenceNH₂-GRKKRRQRRR-COOH (L-TAT (s1a))

SEQ ID NO: 17

or the amino acid sequence NH₂—RKKRRQRRR-COOH (L-TAT (s1b))

SEQ ID NO: 18

.

The person skilled in the art will understand that phrases like that asequence according to generic formula (I) or according to any ofsubformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If) may be used with or beapplied to a particular (trafficking) sequence or may form a basis for atransporter peptide construct to generic formula (I) or any ofsubformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), etc. is intended toillustrate that a sequence is claimed which exhibits certaincharacterisitics with regard to:

-   -   i) the sequence of side chain residues characterizing specific        amino acid entities, and    -   ii) the sequence of D- and L-amino acids in said sequence.

To give an illustrative example: If subformula (If) is used with orapplied to TAT (1-36) (SEQ ID NO:8), the sequence of the side chainresidues (i) is as indicated in SEQ ID NO: 8. However, this claimedsequence is not a pure L-amino acid sequence, but comprises somewherethe motif of subformula (If). Art example for such embodiment would bethe following sequence (D-amino acids indicated in small letters, Lamino acids indicated in capital letters):

MEPVDPRLEP WKHPGSQPKT ACTNCYCKKC CFHCQVCFITKALGISYGrK KRrQRRrPPQ GSQTHQVSLS KQPTSQSRGD PTGPKE.

Since SEQ ID NO:8 comprises 86 amino acids, there are of course severalfurther possibilities of placing the motif of subformula (If) elsewherein this sequence. It is also envisioned that the sequence may comprisein particular embodiments more than one motif of the generic formulaand/or subformulas.

Particular preferred examples of trafficking sequences forming a basisfor a transporter peptide construct to generic formula (I) or any ofsubformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), as defined above, maybe selected, without being limited thereto, from sequences or a partthereof as defined according to Table 1 below, or any fragment orvariant or derivative thereof (as long as it retains the function oftranslocating across a biological membrane).

TABLE 1 SEQUENCE/ SEQ PEPTIDE ID NAME NO AA SEQUENCE TAT (1-86) 8 86MEPVDPRLEP WKHPGSQPKT ACTNCYCKKC CFHCQVCFIT KALGISYGRK KRRQRRRPPQGSQTHQVSLS KQPTSQSRGD PTGPKE TAT (37-72) 9 36 CFITKALGIS YGRKKRRQRRRPPQGSQTHQ VSLSKQ TAT (37-58) 10 22 CFITKALGIS YGRKKRRQRR RPTAT (38-58)GGC 11 24 FITKALGISY GRKKRRQRRR PGGC TAT CGG(47-58) 12 15CGGYGRKKRR QRRRP TAT (47-58)GGC 13 15 YGRKKRRQRR RPGGC TAT (1-72) Mut 1456 MEPVDPRLEP WKHPGSQPKT Cys/Ala 72 AFITKALGIS YGRKKRRQRRRPPQGSQTHQ VSLSKQ L-generic-TAT 15 17 XXXXRKKRRQRRRXXXX(NH₂-XXXXRKKRRQRRRXXXX-COOH) L-generic- 16 11 NH₂-X_(n)^(b)-RKKRRQRRR-X_(n) ^(b)-COOH TAT (s) L-TAT (s1a) 17 10 GRKKRRQRRR(NH₂-GRKKRRQRRR-COOH) L-TAT (s1b) 18 9 RKKRRQRRR (NH₂-GRKKRRQRRR-COOH)L-TAT (s1c) 19 11 YDRKKRRQRRR r₃-L-TAT 20 9 rKKRrQRRr r₃-L-TATi 21 9rRRQrRKKr βA-r₃-L-TAT 22 9 βA-rKKRrQRRr βA-r₃-L-TATi 23 9 βA-rRRQrRKKrFITC-βA-r₃-L- 24 9 FITC-βA-rKKRrQRRr TAT FITC-βA-r₃-L- 25 9FITC-βA-rRRQrRKKr TATi TAT(s2-1) 26 9 rAKRrQRRr TAT(s2-2) 27 9 rKARrQRRrTAT(s2-3) 28 9 rKKArQRRr TAT(s2-4) 29 9 rKKRrARRr TAT(s2-5) 30 9rKKRrQARr TAT(s2-6) 31 9 rKKRrQRAr TAT(s2-7) 32 9 rDKRrQRRr TAT(s2-8) 339 rKDRrQRRr TAT(s2-9) 34 9 rKKDrQRRr TAT(s2-10) 35 9 rKKRrDRRrTAT(s2-11) 36 9 rKKRrQDRr TAT(s2-12) 37 9 rKKRrQRDr TAT(s2-13) 38 9rEKRrQRRr TAT(s2-14) 39 9 rKERrQRRr TAT(s2-15) 40 9 rKKErQRRr TAT(s2-16)41 9 rKKRrERRr TAT(s2-17) 42 9 rKKRrQERr TAT(s2-18) 43 9 rKKRrQRErTAT(s2-19) 44 9 rFKRrQRRr TAT(s2-20) 45 9 rKFRrQRRr TAT(s2-21) 46 9rKKFrQRRr TAT(s2-22) 47 9 rKKRrFRRr TAT(s2-23) 48 9 rKKRrQFRr TAT(s2-24)49 9 rKKRrQRFr TAT(s2-25) 50 9 rRKRrQRRr TAT(s2-26) 51 9 rKRRrQRRrTAT(s2-27) 52 9 rKKKrQRRr TAT(s2-28) 53 9 rKKRrRRRr TAT(s2-29) 54 9rKKRrQKRr TAT(s2-30) 55 9 rKKRrQRKr TAT(s2-31) 56 9 rHKRrQRRr TAT(s2-32)57 9 rKHRrQRRr TAT(s2-33) 58 9 rKKHrQRRr TAT(s2-34) 59 9 rKKRrHRRrTAT(s2-35) 60 9 rKKRrQHRr TAT(s2-36) 61 9 rKKRrQRHr TAT(s2-37) 62 9rIKRrQRRr TAT(s2-38) 63 9 rKIRrQRRr TAT(s2-39) 64 9 rKKIrQRRr TAT(s2-40)65 9 rKKRrIRRr TAT(s2-41) 66 9 rKKRrQIRr TAT(s2-42) 67 9 rKKRrQRIrTAT(s2-43) 68 9 rLKRrQRRr TAT(s2-44) 69 9 rKLRrQRRr TAT(s2-45) 70 9rKKLrQRRr TAT(s2-46) 71 9 rKKRrLRRr TAT(s2-47) 72 9 rKKRrQLRr TAT(s2-48)73 9 rKKRrQRLr TAT(s2-49) 74 9 rMKRrQRRr TAT(s2-50) 75 9 rKMRrQRRrTAT(s2-51) 76 9 rKKMrQRRr TAT(s2-52) 77 9 rKKRrMRRr TAT(s2-53) 78 9rKKRrQMRr TAT(s2-54) 79 9 rKKRrQRMr TAT(s2-55) 80 9 rNKRrQRRr TAT(s2-56)81 9 rKNRrQRRr TAT(s2-57) 82 9 rKKNrQRRr TAT(s2-58) 83 9 rKKRrNRRrTAT(s2-59) 84 9 rKKRrQNRr TAT(s2-60) 85 9 rKKRrQRNr TAT(s2-61) 86 9rQKRrQRRr TAT(s2-62) 87 9 rKQRrQRRr TAT(s2-63) 88 9 rKKQrQRRr TAT(s2-64)89 9 rKKRrKRRr TAT(s2-65) 90 9 rKKRrQQRr TAT(s2-66) 91 9 rKKRrQRQrTAT(s2-67) 92 9 rSKRrQRRr TAT(s2-68) 93 9 rKSRrQRRr TAT(s2-69) 94 9rKKSrQRRr TAT(s2-70) 95 9 rKKRrSRRr TAT(s2-71) 96 9 rKKRrQSRr TAT(s2-72)97 9 rKKRrQRSr TAT(s2-73) 98 9 rTKRrQRRr TAT(s2-74) 99 9 rKTRrQRRrTAT(s2-75) 100 9 rKKTrQRRr TAT(s2-76) 101 9 rKKRrTRRr TAT(s2-77) 102 9rKKRrQTRr TAT(s2-78) 103 9 rKKRrQRTr TAT(s2-79) 104 9 rVKRrQRRrTAT(s2-80) 105 9 rKVRrQRRr TAT(s2-81) 106 9 rKKVrQRRr TAT(s2-82) 107 9rKKRrVRRr TAT(s2-83) 108 9 rKKRrQVRr TAT(s2-84) 109 9 rKKRrQRVrTAT(s2-85) 110 9 rWKRrQRRr TAT(s2-86) 111 9 rKWRrQRRr TAT(s2-87) 112 9rKKWrQRRr TAT(s2-88) 113 9 rKKRrWRRr TAT(s2-89) 114 9 rKKRrQWRrTAT(s2-90) 115 9 rKKRrQRWr TAT(s2-91) 116 9 rYKRrQRRr TAT(s2-92) 117 9rKYRrQRRr TAT(s2-93) 118 9 rKKYrQRRr TAT(s2-94) 119 9 rKKRrYRRrTAT(s2-95) 120 9 rKKRrQYRr TAT(s2-96) 121 9 rKKRrQRYr r₃R₆ 122 9rRRRrRRRr L-R₉ 123 9 RRRRRRRRR L-R₈ 124 8 RRRRRRRR L-R₇ 125 7 RRRRRRRL-R₆ 126 6 RRRRRR L-R₅ 127 5 RRRRR PTD-4 128 11 YARAAARQARA PTD-4 129 11WARAAARQARA (variant 1) PTD-4 130 11 WARAQRAAARA (variant 2)L-P1 Penetratin 131 16 RQVKVWFQNRRMKWKK D-P1 Penetratin 132 16KKWKMRRNQFWVKVQR JNKI, bestfit 133 17 WKRAAARKARAMSLNLF JNKI, bestfit134 17 WKRAAARAARAMSLNLF (variant 1) MDCK transcytose 135 9 RYRGDLGRRsequence YKGL 136 4 YKGL r3 (generic) 252 9 rXXXrXXXr

Particular examples of trafficking sequences forming a basis for atransporter construct according to generic formula (I) or according toany of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), as definedabove, may also be selected from sequences as mentioned herein, e.g. asshown in Table 1, which exhibit the reverse sequence of this specificsequence, i.e., wherein the order of amino acids in the sequence from N-to C-terminal end is reversed.

Particular preferred examples of trafficking sequences forming a basisfor a transporter construct according to generic formula (I) oraccording to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If),as defined above, may be selected from fragments or variants of theabove sequences (with the proviso that such fragment or variant retainthe function to provide for translocation across biological membranes).In this specific context, variants and/or fragments of those traffickingsequences preferably comprise or consist of a peptide sequence having atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 85%, preferably at least90%, more preferably at least 95% and most preferably at least 99% overthe whole length of the native sequence of such a trafficking sequenceas defined above. Additionally, a fragment of such a traffickingsequence may furthermore comprise epitopes (also called “antigendeterminants”) of the full-length trafficking sequence. Epitopes in thecontext of the present invention are typically fragments located on theouter surface of a (native) protein or peptide sequence as definedherein, preferably having 5 to 15 amino acids, more preferably having 5to 12 amino acids, even more preferably having 6 to 9 amino acids, whichmay be recognized by antibodies, i.e. in their native form.

In this specific context, a “fragment” of a trafficking sequence asdefined above, particularly in Table 1 or 3, is preferably to beunderstood as a truncated sequence thereof, i.e. an amino acid sequence,which is N-terminally, C-terminally and/or intrasequentially truncatedcompared to the amino acid sequence of the native sequence.

Furthermore, in the specific context of the present invention, a“variant” of a trafficking sequence or its fragment as defined above,particularly as defined in Table 1 or 3, is preferably to be understoodas a sequence wherein the amino acid sequence of the variant differsfrom the native trafficking sequence or a fragment thereof as definedherein in one or more mutation(s), such as one or more substituted, (or,if necessary, inserted and/or deleted) amino acid(s). Preferably,variants of such a trafficking sequence as defined above have the samebiological function or specific activity compared to the full-lengthnative sequence, i.e. transport into cells and the nucleus. Morepreferably, a variant of such a trafficking sequence as defined abovemay comprise about 1 to 50, 1 to 20, even more preferably 1 to 10 andmost preferably 1 to 5, 4, 3, 2 or 1 amino acid alterations within theabove meaning. Variants of such a trafficking sequence as defined abovemay also comprise conservative amino acid substitutions. Conservativeamino acid substitutions may include synonymous amino acid residueswithin a group which have sufficiently similar physicochemicalproperties, so that a substitution between members of the group willpreserve the biological activity of the molecule (see e.g. Grantham, R.(1974), Science 185, 862-864). It is evident to the skilled person thatamino acids may also be inserted and/or deleted in the above-definedsequences without altering their function, particularly if theinsertions and/or deletions only involve a few amino acids, e.g. lessthan twenty, and preferably less than ten, and do not remove or displaceamino acids which are critical to functional activity. Particularly,conservative amino acid substitutions are preferably substitutions inwhich the amino acids, which originate from the same class of aminoacids (basic amino acids, acidic amino acids, polar amino acids, etc.),are exchanged for one another. In particular, these are amino acids,aliphatic side chains, positively or negatively charged side chains,aromatic groups in the side chains of amino acids, the side chains ofwhich can enter into hydrogen bridges, e.g. side chains which have ahydroxyl function. This means that e.g. an amino acid having a polarside chain is replaced by another amino acid having a likewise polarside chain, or, for example, an amino acid characterized by ahydrophobic side chain is substituted by another amino acid having alikewise hydrophobic side chain (e.g. serine (threonine) by threonine(serine) or leucine (isoleucine) by isoleucine (leucine)). Preferably,synonymous amino acid residues, which are classified into the samegroups and are typically exchangeable by conservative amino acidsubstitutions, are defined in Table 2.

TABLE 2 Preferred Groups of Synonymous Amino Acid Residues Amino AcidSynonymous Residue Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, HisLeu Ile, Phe, Tyr, Met, Val, Leu Pro Gly, Ala, (Thr), Pro ThrPro, Ser, Ala, Gly, His, Gln, Thr Ala Gly, Thr, Pro, Ala ValMet, Tyr, Phe, Ile, Leu, Val Gly Ala, (Thr), Pro, Ser, Gly IleMet, Tyr, Phe, Val, Leu, Ile Phe Trp, Met, Tyr, Ile, Val, Leu, Phe TyrTrp, Met, Phe, Ile, Val, Leu, Tyr Cys Ser, Thr, Cys HisGlu, Lys, Gln, Thr, Arg, His Gln Glu, Lys, Asn, His, (Thr), Arg, Gln AsnGln, Asp, Ser, Asn Lys Glu, Gln, His, Arg, Lys Asp Glu, Asn, Asp GluAsp, Lys, Asn, Gln, His, Arg, Glu Met Phe, Ile, Val, Leu, Met Trp Trp

The length of a trafficking sequences used for a transporter constructas defined above, i.e. the number of contiguous amino acids selectedfrom any of the trafficking sequences as defined above, may in aparticular embodiment be determined by the number of amino acids ofgeneric formula (I) or by any of subformulas (Ia), (Ib), (Ic), (Id),(Ie), or (If) as defined above. The (sequence) length of an inventivenovel transporter construct according to generic formula (I) oraccording to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If),may therefore deviate from the length of the exemplary traffickingsequences as shown herein, e.g. in Table 1 or 3 or as generally definedherein. In other words, the length of the transporter constructaccording to generic formula (I) or according to any of subformulas(Ia), (Ib), (Ic), (Id), (Ie), or (If) as defined above may determine thelength of the amino acid sequence, which may be taken from a traffickingsequence as defined herein, provided that the amino acid sequence istaken from a contiguous stretch of amino acids of said traffickingsequence. As an example, if the length of a transporter constructaccording to generic formula (I) or according to any of subformulas(Ia), (Ib), (Ic), (Id), (Ie), or (If), as defined above, is 9 aminoacids, a sequence suitable as a transporter construct is derived from 9contiguous amino acids of a trafficking sequence as defined above,wherein the selected amino acid sequence may be derived from anyposition or region of said trafficking sequence. The same holds for anyother length determined by generic formula (I) or by any of subformulas(Ia), (Ib), (Ic), (Id), (Ie), or (If), as defined above. It is alsocontemplated that in some embodiments the trafficking sequence of thetransporter construct according to the present invention is less than150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, and/orless than 10 amino acids in length.

According to another preferred embodiment, an inventive noveltransporter construct according to generic formula (I) or according toany of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), comprises orconsists of at least one variant and/or fragment of the above definedsequences. Preferably, these variants and/or fragments retain biologicalactivity of the inventive novel transporter constructs according togeneric formula (I) or according to any of subformulas (Ia), (Ib), (Ic),(Id), (Ie), or (If), as disclosed above. Functionality of such fragmentsor variants may be tested by various tests, e.g. transfection efficacy,correct expression of proteins encoded by cargo nucleic acids, or bybiophysical methods, e.g. spectroscopy, computer modeling, structuralanalysis, etc. Particularly, the inventive novel transporter constructsaccording to generic formula (I) or according to any of subformulas(Ia), (Ib), (Ic), (Id), (Ie), or (If), as disclosed above or variantsand/or fragments thereof may be analyzed by hydrophilicity analysis (seee.g. Hopp and Woods, 1981. Proc Natl Acad Sci USA 78: 3824-3828) thatcan be utilized to identify the hydrophobic and hydrophilic regions ofthe peptides, thus aiding in the design of substrates for experimentalmanipulation. Secondary structural analysis may also be performed toidentify regions of the inventive novel transporter construct accordingto generic formula (I) or according to any of subformulas (Ia), (Ib),(Ic), (Id), (Ie), or (If), as disclosed above or of variants and/orfragments thereof that assume specific structural motifs (see e.g. Chouand Fasman, 1974, Biochem 13: 222-223). Manipulation, translation,secondary structure prediction, hydrophilicity and hydrophobicityprofiles, open reading frame prediction and plotting, and determinationof sequence homologies can be accomplished using computer softwareprograms available in the art. Other methods of structural analysisinclude, e.g. X-ray crystallography (see e.g. Engstrom, 1974. BiochemExp Biol 11: 7-13), mass spectroscopy and gas chromatography (see e.g.METHODS IN PROTEIN SCIENCE, 1997, J. Wiley and Sons, New York, N.Y.) andcomputer modeling (see e.g. Fletterick and Zoller, eds., 1986. ComputerGraphics and Molecular Modeling, In: CURRENT COMMUNICATIONS IN MOLECULARBIOLOGY, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)may also be employed.

Likewise, the inventive novel transporter construct according to genericformula (I) or according to any of subformulas (Ia), (Ib), (Ic), (Id),(Ie), or (If) above, comprises or consists of at least one variant(and/or fragment) of the above defined transporter constructs. In thecontext of the invention variants (and/or fragments) of these noveltransporter constructs may have a sequence identity to their nativetransporter construct according to generic formula (I) or according toany of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If) of at least70%, 80% or 85%, preferably at least 90%, more preferably at least 95%and most preferably at least 99% over the whole length of the nativetransporter construct as defined above.

A “fragment” of an inventive novel transporter construct according togeneric formula (I) or according to any of subformulas (Ia), (Ib), (Ic),(Id), (Ie), or (If) above, is preferably to be understood as a truncatedsequence thereof, i.e. an amino acid sequence of the novel transporterconstruct, which is N-terminally, C-terminally and/or intrasequentiallytruncated compared to the amino acid sequence of the native sequence,e.g. the native inventive novel transporter construct according togeneric formula (I) or according to any of subformulas (Ia), (Ib), (Ic),(Id), (Ie), or (If) above.

A “variant” of an inventive novel transporter construct according togeneric formula (I) or according to any of subformulas (Ia), (Ib), (Ic),(Id), (Ie), or (If) above preferably comprises a sequence wherein theamino acid sequence of the transporter construct variant differs fromthe native sequence of the transporter construct as defined herein inone or more mutation(s), such as one or more substituted, (or, ifnecessary, inserted and/or deleted) amino acid(s). Preferably, variantsof such inventive transporter constructs have the same biologicalfunction or specific activity compared to the full-length nativeinventive transporter constructs as defined above. Preferably, a variantof inventive transporter constructs may comprise about 1 to 50, 1 to 20,preferably 1 to 10 and more preferably 1 to 5, 1 to 4, 1 to 3, 1 to 2 or1 amino acid alteration(s) within the above meaning. Such alterationsmay comprise inter alia modifications of amino acids as defined above,introduction of labels into amino acids as defined above, substitutingan amino acid with any of the (modified or labelled) amino acidsmentioned herein, deletions or insertions of amino acids. Variants asdefined herein furthermore preferably comprise conservative amino acidsubstitutions, preferably such as already defined above.

In order to determine the percentage to which two amino acid sequencesare identical, particularly the amino acid sequence of an inventivenovel transporter construct according to generic formula (I) oraccording to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If)above, or of any further amino acid sequence as defined herein, theamino acid sequences can be aligned in order to be subsequently comparedto one another. Therefore, as an example, e.g. gaps can be inserted intothe sequence of the first amino acid sequence and the component at thecorresponding position of the second amino acid sequence can becompared. If a position in the first amino acid sequence is occupied bythe same component as is the case at a position in the second amino acidsequence, the two sequences are identical at this position. Thepercentage to which two sequences are identical is a function of thenumber of identical positions divided by the total number of positions.The same, of course also applies accordingly to nucleic acid sequences.In the above context, an amino acid sequence having a sequence “sharinga sequence identity” of at least, for example, 95% to a query amino acidsequence of the present invention, is intended to mean that the sequenceof the subject amino acid sequence is identical to the query sequenceexcept that the subject amino acid sequence may include up to five aminoacid alterations per each 100 amino acids of the query amino acidsequence. In other words, to obtain an amino acid sequence having asequence of at least 95% identity to a query amino acid sequence, up to5% (5 of 100) of the amino acid residues in the subject sequence may beinserted or substituted with another amino acid or deleted, preferablywithin the above definitions of variants or fragments. The same, ofcourse, also applies similarly to nucleic acid sequences.

For (amino acid or nucleic acid) sequences without exact correspondence,a “% identity” of a first sequence may be determined with respect to asecond sequence. In general, these two sequences to be compared arealigned to give a maximum correlation between the sequences. This mayinclude inserting “gaps” in either one or both sequences, to enhance thedegree of alignment. A % identity may then be determined over the wholelength of each of the sequences being compared (so-called globalalignment), that is particularly suitable for sequences of the same orsimilar length, or over shorter, defined lengths (so-called localalignment), that is more suitable for sequences of unequal length.

Methods for comparing the identity and homology of two or more sequencesare well known in the art. The percentage to which two sequences areidentical can e.g. be determined using a mathematical algorithm. Apreferred, but not limiting, example of a mathematical algorithm whichcan be used is the algorithm of Karlin et al. (1993), PNAS USA,90:5873-5877. Such an algorithm is integrated in the BLAST family ofprograms, e.g. BLAST or NBLAST program (see also Altschul et al., 1990,J. Mol. Biol. 215, 403-410 or Altschul et al. (1997), Nucleic Acids Res,25:3389-3402), accessible through the home page of the NCBI at worldwide web site ncbi.nlm.nih.gov) and FASTA (Pearson (1990), MethodsEnzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci.U.S.A 85, 2444-2448). Sequences which are identical to other sequencesto a certain extent can be identified by these programmes. Furthermore,programs available in the Wisconsin Sequence Analysis Package, version9.1 (Devereux et al., 1984, Nucleic Acids Res., 387-395), for examplethe programs BESTFIT and GAP, may be used to determine the % identitybetween two polynucleotides and the % identity and the % homology oridentity between two polypeptide sequences. BESTFIT uses the “localhomology” algorithm of (Smith and Waterman (1981), J. Mol. Biol. 147,195-197) and finds the best single region of similarity between twosequences.

According to a particularly preferred embodiment the inventive noveltransporter construct as defined above comprises the subformula (If)DLLLDLLLD (SEQ ID NO: 7) as defined above, wherein the transportersequence is selected from any of the above mentioned (specific)sequences, even more preferably from following sequences:

TABLE 3 SEQUENCE/ SEQ PEPTIDE ID NAME NO AA SEQUENCE r₃-L-TAT 20 9rKKRrQRRr r₃-L-TATi 21 9 rRRQrRKKr βA-r₃-L-TAT 22 9 βA-rKKRrQRRrβA-r₃-L-TATi 23 9 βA-rRRQrRKKr FITC-βA-r₃-L-TAT 24 9 FITC-βA-rKKRrQRRrFITC-βA-r₃-L-TATi 25 9 FITC-βA-rRRQrRKKr TAT(s2-1) 26 9 rAKRrQRRrTAT(s2-2) 27 9 rKARrQRRr TAT(s2-3) 28 9 rKKArQRRr TAT(s2-4) 29 9rKKRrARRr TAT(s2-5) 30 9 rKKRrQARr TAT(s2-6) 31 9 rKKRrQRAr TAT(s2-7) 329 rDKRrQRRr TAT(s2-8) 33 9 rKDRrQRRr TAT(s2-9) 34 9 rKKDrQRRr TAT(s2-10)35 9 rKKRrDRRr TAT(s2-11) 36 9 rKKRrQDRr TAT(s2-12) 37 9 rKKRrQRDrTAT(s2-13) 38 9 rEKRrQRRr TAT(s2-14) 39 9 rKERrQRRr TAT(s2-15) 40 9rKKErQRRr TAT(s2-16) 41 9 rKKRrERRr TAT(s2-17) 42 9 rKKRrQERr TAT(s2-18)43 9 rKKRrQREr TAT(s2-19) 44 9 rFKRrQRRr TAT(s2-20) 45 9 rKFRrQRRrTAT(s2-21) 46 9 rKKFrQRRr TAT(s2-22) 47 9 rKKRrFRRr TAT(s2-23) 48 9rKKRrQFRr TAT(s2-24) 49 9 rKKRrQRFr TAT(s2-25) 50 9 rRKRrQRRr TAT(s2-26)51 9 rKRRrQRRr TAT(s2-27) 52 9 rKKKrQRRr TAT(s2-28) 53 9 rKKRrRRRrTAT(s2-29) 54 9 rKKRrQKRr TAT(s2-30) 55 9 rKKRrQRKr TAT(s2-31) 56 9rHKRrQRRr TAT(s2-32) 57 9 rKHRrQRRr TAT(s2-33) 58 9 rKKHrQRRr TAT(s2-34)59 9 rKKRrHRRr TAT(s2-35) 60 9 rKKRrQHRr TAT(s2-36) 61 9 rKKRrQRHrTAT(s2-37) 62 9 rIKRrQRRr TAT(s2-38) 63 9 rKIRrQRRr TAT(s2-39) 64 9rKKIrQRRr TAT(s2-40) 65 9 rKKRrIRRr TAT(s2-41) 66 9 rKKRrQIRr TAT(s2-42)67 9 rKKRrQRIr TAT(s2-43) 68 9 rLKRrQRRr TAT(s2-44) 69 9 rKLRrQRRrTAT(s2-45) 70 9 rKKLrQRRr TAT(s2-46) 71 9 rKKRrLRRr TAT(s2-47) 72 9rKKRrQLRr TAT(s2-48) 73 9 rKKRrQRLr TAT(s2-49) 74 9 rMKRrQRRr TAT(s2-50)75 9 rKMRrQRRr TAT(s2-51) 76 9 rKKMrQRRr TAT(s2-52) 77 9 rKKRrMRRrTAT(s2-53) 78 9 rKKRrQMRr TAT(s2-54) 79 9 rKKRrQRMr TAT(s2-55) 80 9rNKRrQRRr TAT(s2-56) 81 9 rKNRrQRRr TAT(s2-57) 82 9 rKKNrQRRr TAT(s2-58)83 9 rKKRrNRRr TAT(s2-59) 84 9 rKKRrQNRr TAT(s2-60) 85 9 rKKRrQRNrTAT(s2-61) 86 9 rQKRrQRRr TAT(s2-62) 87 9 rKQRrQRRr TAT(s2-63) 88 9rKKQrQRRr TAT(s2-64) 89 9 rKKRrKRRr TAT(s2-65) 90 9 rKKRrQQRr TAT(s2-66)91 9 rKKRrQRQr TAT(s2-67) 92 9 rSKRrQRRr TAT(s2-68) 93 9 rKSRrQRRrTAT(s2-69) 94 9 rKKSrQRRr TAT(s2-70) 95 9 rKKRrSRRr TAT(s2-71) 96 9rKKRrQSRr TAT(s2-72) 97 9 rKKRrQRSr TAT(s2-73) 98 9 rTKRrQRRr TAT(s2-74)99 9 rKTRrQRRr TAT(s2-75) 100 9 rKKTrQRRr TAT(s2-76) 101 9 rKKRrTRRrTAT(s2-77) 102 9 rKKRrQTRr TAT(s2-78) 103 9 rKKRrQRTr TAT(s2-79) 104 9rVKRrQRRr TAT(s2-80) 105 9 rKVRrQRRr TAT(s2-81) 106 9 rKKVrQRRrTAT(s2-82) 107 9 rKKRrVRRr TAT(s2-83) 108 9 rKKRrQVRr TAT(s2-84) 109 9rKKRrQRVr TAT(s2-85) 110 9 rWKRrQRRr TAT(s2-86) 111 9 rKWRrQRRrTAT(s2-87) 112 9 rKKWrQRRr TAT(s2-88) 113 9 rKKRrWRRr TAT(s2-89) 114 9rKKRrQWRr TAT(s2-90) 115 9 rKKRrQRWr TAT(s2-91) 116 9 rYKRrQRRrTAT(s2-92) 117 9 rKYRrQRRr TAT(s2-93) 118 9 rKKYrQRRr TAT(s2-94) 119 9rKKRrYRRr TAT(s2-95) 120 9 rKKRrQYRr TAT(s2-96) 121 9 rKKRrQRYr r₃R₆ 1229 rRRRrRRRr L-R₉ 123 9 RRRRRRRRR

In the above table, the subformula (If) DLLLDLLLD (SEQ ID NO: 7) isapplied to the above specific sequences, i.e. novel inventivetransporter construct as defined above comprises 9 amino acids, whereinthe D-enatiomeric amino acids are indicated herein with a smallcharacter and the L-enatiomeric amino acids are indicated with a capitalletter.

In a particular embodiment the inventive transporter contruct compriseor consist of at least one sequence according to rXXXrXXXr (SEQ ID NO:252), wherein:

-   -   r represents an D-enatiomeric arginine;    -   X is am, L-amino acid;        and wherein each X may be selected individually and        independently of any other X within SEQ ID NO: 252. Preferably        at least 4 out of said 6×L-amino acids within SEQ ID NO: 252 are        K or R. In another embodiment the transporter construct        according to the present invention comprises or consists of the        sequence rX₁X₂X₃rX₄X₅X₆r (SEQ ID NO: 252) wherein X₁ is X₂ is K,        X₃ is R and X₄, X₅, and X₆ are any L-amino acid selected        independently from each other. Similarly, the transporter        construct according to the present invention may comprise or        consist of the sequence rX₁X₂X₃rX₄X₅X₆r (SEQ ID NO: 252),        wherein X₄ is Q, X₅ is R, X₆ is R and X₁, X₂, and X₃ are any        L-amino acid selected independently from each other. The        inventive transporter construct may also comprises or consist of        the sequence rX₁X₂X₃rX₄X₅X₆r (SEQ ID NO: 252), wherein one, two,        three, four, five or six X amino acid residues are chosen from        the group consisting of: X₁ is K, X₂ is K, X₃ is R, X₄ is Q, X₅        is R, X₆ is R, while the remaining X amino acid residues not        selected from above group may be any L-amino acid and are        selected independently from each other. X₁ is then preferably Y        and/or X₄ is preferably K or R. Similarly considered are reverse        sequences of the above mentioned sequences and embodiments of        SEQ ID NO: 252.

Surprisingly, the present inventors found, that a tyrosine (Y) atposition 2 of formula (I) above not only significantly increases uptakebut also a significantly increased intracellular concentration afterinoculation for 25 hours. This is particularly observed for transporterconstructs which comprise a trafficking sequence derived from HIV-1 TATprotein as shown in Table 1, which show an Y in position 2, preferablyhave 9 aa and exhibiting the consensus sequence rXXXrXXXr (SEQ ID NO:252). According to a particularly preferred embodiment of the presentinvention, the inventive novel transporter construct as defined abovetherefore comprises transporter constructs of general formula (I)defined above or even more preferably according to subformula (If) asdefined above, wherein the transporter sequence is used with or isapplied to a trafficking sequence derived from HIV-1 TAT protein havinga Tyrosine (Y) at position 2 of the TAT derived sequence. Even morepreferably, the transporter sequence is used with or is applied to atrafficking sequence derived from HIV-1 TAT protein having a Tyrosine(Y) at position 2 of the TAT derived sequence, wherein the transporterconstruct of general formula (I) defined above or according tosubformula (If) as defined above preferably has 9 aa and the consensussequence rXXXrXXXr (SEQ ID NO: 252 wherein the D-enatiomeric amino acidsare indicated herein with a small character and the L-enatiomeric aminoacids are indicated with a capital letter). Most preferably, thetransporter sequence is used with or is applied to a traffickingsequence derived from HIV-1 TAT, wherein the transporter construct ofgeneral formula (I) defined above or according to subformula (If) asdefined above preferably has 9 aa and the consensus sequence rXXXrXXXr(SEQ ID NO: 252) and the trafficking sequence is selected from thesequence TAT(s2-91) (SEQ ID NO: 116).

The inventive novel transporter constructs according to generic formula(I) or according to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or(If) above, may furthermore comprise at least one modification,preferably at their termini, either at the C- or the N-terminus or both.The C-Terminus may preferably be modified by an amide modification,whereas the N-terminus may be modified by any suitable NFL-protectiongroup, such as e.g. acylation, or any further modification as alreadyindicated above for L-amino acids and D-amino acids. Such modificationsalso includes introduction of labels as defined above.

The inventive novel transporter constructs according to generic formula(I) or according to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or(If) above, may be obtained or produced by methods well-known in theart, e.g. by chemical synthesis as defined above or by geneticengineering methods.

According to a second aspect of the present invention the underlyingobject is solved by an inventive transporter cargo conjugate molecule,comprising as a component (A) the inventive novel transporter constructaccording to generic formula (I) or according to any of subformulas(Ia), (Ib), (Ic), (Id), (Ie), or (If) above, and as a component (B) aneffector molecule, e.g. selected from proteins or peptides, such astherapeutically active proteins and peptides, protein kinase inhibitors,particularly inhibitors of the protein kinase c-Jun amino terminalkinase or factors, antigens, antibodies, apoptotic factors, proteasesimplicated in pathological states, preferably peptidic proteaseinhibitors, BH3-domains BH3-only proteins, or selected from nucleicacids, siRNAs, antisense RNAs or from cytotoxic agents, small organiccompounds, etc.

In the context of the present invention, a therapeutically activeprotein or peptide suitable as the effector molecule for component (B)of the inventive transporter cargo conjugate molecule may be selectedfrom, without being limited thereto, proteins, capable of stimulating orinhibiting the signal transduction in the cell, e.g. cytokines,antibodies, etc. Therapeutically active proteins may thus comprisecytokines of class I of the family of cytokines, having 4 positionallyconserved cysteine residues (CCCC) and comprising a conserved sequencemotif Trp-Ser-X-Trp-Ser (WSXWS; SEQ ID NO: 253), wherein X is anon-conserved amino acid. Cytokines of class I of the family ofcytokines comprise the GM-CSF subfamily, e.g. IL-3, IL-5, GM-CSF, theIL-6-subfamily, e.g. IL-6, IL-12, or the IL-2-subfamily, e.g. IL-2,IL-4, IL-7, IL-9, IL-15, etc., or the cytokines IL-1alpha, IL-1beta,IL-10 etc. Therapeutically active proteins may also comprise cytokinesof class II of the family of cytokines, which also comprise 4positionally conserved cystein residues (CCCC; SEQ ID NO: 254), but noconserved sequence motif Trp-Ser-X-Trp-Ser (WSXWS; SEQ ID NO: Cytokinesof class II of the family of cytokines comprise e.g. IFN-alpha,IFN-beta, IFN-gamma, etc. Therapeutically active proteins mayadditionally comprise cytokines of the family of tumor necrose factors,e.g. TNF-alpha, TNF-beta, etc., or cytokines of the family ofchemokines, which comprise 7 transmembrane helices and interact withG-protein, e.g. IL-8, MIP-1, RANTES, CCR5, CXR4, etc., or cytokinespecific receptors, such as TNF-RI, TNF-RII, CD40, OX40 (CD134), Fas, orfrom fragments or variants thereof. Preferably, such fragments as wellas variants exhibit a sequence homology or identity of about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,or about 90% with one of the protein or peptide sequences as shown ordescribed above. In this context, fragments and variants are preferablyas defined above for component (A) of the inventive transporter cargoconjugate molecule.

Therapeutically active proteins suitable as the effector molecule ofcomponent (B) of the inventive transporter cargo conjugate molecule mayalso be selected from any of the proteins given in the following: oATL3,oFC3, oPA3, oPD2, 4-1BBL, 5T4, 6Ckine, 707-AP, 9D7, A2M, AA, AAAS, AACT,AASS, ABAT, ABCA1, ABCA4, ABCB1, ABCB11, ABCB2, ABCB4, ABCB7, ABCC2,ABCC6, ABCC8, ABCD1, ABCD3, ABCG5, ABCG8, ABL1, ABO, ABR ACAA1, ACACA,ACADL, ACADM, ACADS, ACADVL, ACAT1, ACCPN, ACE, ACHE, ACHM3, ACHM1,ACLS, ACPI, ACTA1, ACTC, ACTN4, ACVRL1, AD2, ADA, ADAMTS13, ADAMTS2,ADFN, ADH1B, ADH1C, ADLDH3A2, ADRB2, ADRB3, ADSL, AEZ, AFA, AFD1, AFP,AGA, AGL, AGMX2, AGPS, AGS1, AGT, AGTR1, AGXT, AH02, AHCY, AHDS, AHHR,AHSG, AIC, AIED, AIH2, AIH3, AIM-2, AIPL1, AIRE, AK1, ALAD, ALAS2, ALB,HPG1, ALDH2, ALDH3A2, ALDH4A1, ALDH5A1, ALDH1A1, ALDOA, ALDOB, ALMS1,ALPL, ALPP, ALS2, ALX4, AMACR, AMBP, AMCD, AMCD1, AMCN, AMELX, AMELY,AMGL, AMH, AMHR2, AMPD3, AMPD1, AMT, ANC, ANCR, ANK1, ANOP1, AOM, AP0A4,AP0C2, AP0C3, AP3B1, APC, aPKC, APOA2, APOA1, APOB, APOC3, APOC2, APOE,APOH, APP, APRT, APS1, AQP2, AR, ARAF1, ARG1, ARHGEF12, ARMET, ARSA,ARSB, ARSC2, ARSE, ART-4, ARTC1/m, ARTS, ARVD1, ARX, AS, ASAH, ASAT,ASD1, ASL, ASMD, ASMT, ASNS, ASPA, ASS, ASSP2, ASSP5, ASSP6, AT3, ATD,ATHS, ATM, ATP2A1, ATP2A2, ATP2C1, ATP6B1, ATP7A, ATP7B, ATP8B1, ATPSK2,ATRX, ATXN1, ATXN2, ATXN3, AUTS1, AVMD, AVP, AVPR2, AVSD1, AXIN1, AXIN2,AZF2, B2M, B4GALT7, B7H4, BAGE, BAGE-1, BAX, BBS2, BBS3, BBS4, BCA225,BCAA, BCH, BCHE, BCKDHA, BCKDHB, BCL10, BCL2, BCL3, BCL5, BCL6, BCPM,BCR, BCR/ABL, BDC, BDE, BDMF, BDMR, BEST1, beta-Catenin/m, BF, BFHD,BFIC, BFLS, BFSP2, BGLAP, BGN, BHD, BHR1, BING-4, BIRC5, BJS, BLM, BLMH,BLNK, BMPR2, BPGM, BRAF, BRCA1, BRCA1/m, BRCA2, BRCA2/m, BRCD2, BRCD1,BRDT, BSCL, BSCL2, BTAA, BTD, BTK, BUB1, BWS, BZX, CoL2A1, CoL6A1, C1NH,C1QA, C1QB, C1QG, C1S, C2, C3, C4A, C4B, C5, C6, C7, C7orf2, C8A, C8B,C9, CA125, CA15-3/CA 27-29, CA195, CA19-9, CA72-4, CA2, CA242, CA50,CABYR, CACD, CACNA2D1, CACNA1A, CACNA1F, CACNA1S, CACNB2, CACNB4, CAGE,CA1, CALB3, CALCA, CALCR, CALM, CALR, CAM43, CAMEL, CAP-1, CAPN3,CARD15, CASP-5/m, CASP-8, CASP-8/m, CASR, CAT, CATM, CAV3, CB1, CBBM,CBS, CCA1, CCAL2, CCAL1, CCAT, CCL-1, CCL-11, CCL-12, CCL-13, CCL-14,CCL-15, CCL-16, CCL-17, CCL-18, CCL-19, CCL-2, CCL-20, CCL-21, CCL-22,CCL-23, CCL-24, CCL-25, CCL-27, CCL-3, CCL-4, CCL-5, CCL-7, CCL-8, CCM1,CCNB1, CCND1, CCO, CCR2, CCR5, CCT, CCV, CCZS, CD1, CD19, CD20, CD22,CD25, CD27, CD27L, cD3, CD30, CD30, CD30L, CD33, CD36, CD3E, CD3G, CD3Z,CD4, CD40, CD40L, CD44, CD44v, CD44v6, CD52, CD55, CD56, CD59, CD80,CD86, CDAN1, CDAN2, CDAN3, CDC27, CDC27/m, CDC2L1, CDH1, CDK4, CDK4/m,CDKN1C, CDKN2A, CDKN2A/m, CDKN1A, CDKN1C, CDL1, CDPD1, CDR1, CEA,CEACAM1, CEACAM5, CECR, CECR9, CEPA, CETP, CFNS, CFTR, CGF1, CHAC,CHED2, CHED1, CHEK2, CHM, CHML, CHR39C, CHRNA4, CHRNA1, CHRNB1, CHRNE,CHS, CHS1, CHST6, CHX10, CIAS1, CIDX, CKN1, CLA2, CLA3, CLA1, CLCA2,CLCN1, CLCN5, CLCNKB, CLDN16, CLP, CLN2, CLN3, CLN4, CLN5, CLN6, CLN8,C1QA, C1QB, C1QG, C1R, CLS, CMCWTD, CMDJ, CMD1A, CMD1B, CMH2, MH3, CMH6,CMKBR2, CMKBR5, CML28, CML66, CMM, CMT2B, CMT2D, CMT4A, CMT1A, CMTX2,CMTX3, C-MYC, CNA1, CND, CNGA3, CNGA1, CNGB3, CNSN, CNTF, COA-1/m, COCH,COD2, COD1, COH1, COL10A, COL2A2, COL11A2, COL17A1, COL1A1, COL1A2,COL2A1, COL3A1, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A1,COL6A2, COL6A3, COL7A1, COL8A2, COL9A2, COL9A3, COL11A1, COL1A2,COL23A1, COL1A1, COLQ, COMP, COMT, CORD5, CORD1, COX10, COX-2, CP, CPB2,CPO, CPP, CPS1, CPT2, CPT1A, CPX, CRAT, CRB1, CRBM, CREBBP, CRH, CRHBP,CRS, CRV, CRX, CRYAB, CRYBA1, CRYBB2, CRYGA, CRYGC, CRYGD, CSA, CSE,CSF1R, CSF2RA, CSF2RB, CSF3R, CSF1R, CST3, CSTB, CT, CT7, CT-9/BRD6,CTAA1, CTACK, CTEN, CTH, CTHM, CTLA4, CTM, CTNNB1, CTNS, CTPA, CTSB,CTSC, CTSK, CTSL, CTS1, CUBN, CVD1, CX3CL1, CXCL1, CXCL10, CXCL11,CXCL12, CXCL13, CXCL10, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8,CXCL9, CYB5, CYBA, CYBB, CYBB5, CYFRA21-1, CYLD, CYLD1, CYMD, CYP11B1,CYP11B2, CYP17, CYP17A1, CYP19, CYP19A1, CYP1A2, CYP1B1, CYP21A2,CYP27A1, CYP27B1, CYP2A6, CYP2C, CYP2C19, CYP2C9, CYP2D, CYP2D6,CYP2D7P1, CYP3A4, CYP7B1, CYPB1, CYP11B1, CYP1A1, CYP1B1, CYRAA,D40DAD1, DAM, DAM-10/MAGE-B1, DAM-6/MAGE-B2, DAX1, DAZ, DBA, DBH, DBI,DBT, DCC, DC-CK1, DCK, DCR, DCX, DDB1, DDB2, DDIT3, DDU, DECR1, DEK-CAN,DEM, DES, DF, DFN2, DFN4, DFN6, DFNA4, DFNA5, DFNB5, DGCR, DHCR7, DHFR,DHOF, DHS, DIA1, DIAPH2, DIAPH1, DIH1, DIO1, DISCI, DKC1, DLAT, DLD,DLL3, DLX3, DMBT1, DMD, DM1, DMPK, DMWD, DNAI1, DNASE1, DNMT3B, DPEP1,DPYD, DPYS, DRD2, DRD4, DRPLA, DSCR1, DSG1, DSP, DSPP, DSS, DTDP2, DTR,DURS1, DWS, DYS, DYSF, DYT2, DYT3, DYT4, DYT2, DYT1, DYX1, EBAF, EBM,EBNA, EBP, EBR3, EBS1, ECA1, ECB2, ECE1, ECGF1, ECT, ED2, ED4, EDA,EDAR, ECA1, EDN3, EDNRB, EEC1, EEF1A1L14, EEGV1, EFEMP1, EFTUD2/m, EGFR,EGFR/Her1, EGI, EGR2, EIF2AK3, eIF4G, EKV, EIIS, ELA2, ELF2, ELF2M,ELK1, ELN, ELONG, EMD, EML1, EMMPRIN, EMX2, ENA-78, ENAM, END3, ENG,ENO1, ENPP1, ENUR2, ENUR1, EOS, EP300, EPB41, EPB42, EPCAM, EPD, EphA1,EphA2, EphA3, EphrinA2, EphrinA3, EPHX1, EPM2A, EPO, EPOR, EPX, ERBB2,ERCC2 ERCC3, ERCC4, ERCC5, ERCC6, ERVR, ESR1, ETFA, ETFB, ETFDH, ETM1,ETV6-AML1, ETV1, EVC, EVR2, EVR1, EWSR1, EXT2, EXT3, EXT1, EYA1, EYCL2,EYCL3, EYCL1, EZH2, F10, F11, F12, F13A1, F13B, F2, F5, F5F8D, P7, F8,F8C, F9, FABP2, FACL6, FAH, FANCA, FANCB, FANCC, FANCD2, FANCF, FasL,FBN2, FBN1, FBP1, FCG3RA, FCGR2A, FCGR2B, FCGR3A, FCHL, FCMD, FCP1,FDPSL5, FECH, FEO, FEOM1, FES, FGA, FGB, FGD1, FGF2, FGF23, FGF5, FGFR2,FGFR3, FGFR1, FGG, FGS1, FH, FIC1, FIH, F2, FKBP6, FLNA, FLT4, FMO3,FMO4, FMR2, FMR1, FN, FN1/m, FOXC1, FOXE1, FOXL2, FOXO1A, FPDMM, FPF,Fra-1, FRAXF, FRDA, FSHB, FSHMD1A, FSHR, FTH1, FTHL17, FTL, FTZF1,FUCA1, FUT2, FUT6, FUT1, FY, G250, G250/CAIX, G6PC, G6PD, G6PT1, G6PT2,GAA, GABRA3, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b,GAGE-8, GALC, GALE, GALK1, GALNS, GALT, GAMT, GAN, GAST, GASTRIN17,GATA3, GATA, GBA, GBE, GC, GCDH, GCGR, GCH1, GCK, GCP-2, GCS1, G-CSF,GCSH, GCSL, GCY, GDEP, GDF5, GDI1, GDNF, GDXY, GFAP, GFND, GGCX, GGT1,GH2, GH1, GHR, GHRHR, GHS, GIF, GINGF, GIP, GJA3, GJA8, GJB2, GJB3,GJB6, GJB1, GK, GLA, GLB, GLB1, GLC3B, GLC1B, GLC1C, GLDC, GLI3, GLP1,GLRA1, GLUD1, GM1 (fuc-GM1), GM2A, GM-CSF, GMPR, GNAI2, GNAS, GNAT1,GNB3, GNE, GNPTA, GNRH, GNRH1, GNRHR, GNS, GnT-V, gp100, GP1BA, GP1BB,GP9, GPC3, GPD2, GPDS1, GPI, GP1BA, GPN1LW, GPNMB/m, GPSC, GPX1, GRHPR,GRK1, GROα, GROβ, GROγ, GRPR, GSE, GSM1, GSN, GSR, GSS, GTD, GTS,GUCA1A, GUCY2D, GULOP, GUSB, GUSM, GUST, GYPA, GYPC, GYS1, GYS2, HoKPP2,HoMG2, HADHA, HADHB, HAGE, HAGH, HAL, HAST-2, HB1, HBA2, HBA1, HBB,HBBP1, HBD, HBE1, HBG2, HBG1, HBHR, HBP1, HBQ1, HBZ, HBZP, HCA, HCC-1,HCC-4, HCF2, HCG, HCL2, HCL1, HCR, HCVS, HD, HPN, HER2, HER2/NEU, HER3,HERV-K-MEL, HESX1, HEXA, HEXB, HF1, HFE, HF1, HGD, HHC2, HHC3, HHG, HK1,HLA-A, HLA-A*0201-R170I, HLA-A11/m, HLA-A2/m, HLA-DPB1 HLA-DRA, HLCS,HLXB9, HMBS, HMGA2, HMGCL, HMI, HMN2, HMOX1, HMS1 HMW-MAA, HND, HNE,HNF4A, HOAC, HOMEOBOX NKX 3.1, HOM-TES-14/SCP-1, HOM-TES-85, HOXA1HOXD13, HP, HPC1, HPD, HPE2, HPE1, HPFH, HPFH2, HPRT1, HPS1, HPT,HPV-E6, HPV-E7, HR, HRAS, HRD, HRG, HRPT2, HRPT1, HRX, HSD11B2, HSD17B3,HSD17B4, HSD3B2, HSD3B3, HSN1, HSP70-2M, HSPG2, HST-2, HTC2, HTC1,hTERT, HTN3, HTR2C, HVBS6, HVBS1, HVEC, HV1S, HYAL1, HYR, I-309, IAB,IBGC1, IBM2, ICAM1, ICAM3, iCE, ICHQ, ICR5, ICR1, ICS 1, IDDM2, IDDM1,IDS, IDUA, IF, □IFNa/b, □IFNGR1, IGAD1, IGER, IGF-1R, IGF2R, IGF1, IGH,IGHC, IGHG2, IGHG1, IGHM, IGHR, IGKC, IHG1, IHH, IKBKG, IL1, IL-1 RA,IL10, IL-11, IL12, IL12RB1, IL13, IL-13Rα2, IL-15, IL-16, IL-17, IL18,IL-1a, IL-1α, IL-1β, IL-1β, IL1RAPL1, IL2, IL24, IL-2R, IL2RA, IL2RG,IL3, IL3RA, IL4, IL4R, IL4R, IL-5, IL6, IL-7, IL7R, IL-8, IL-9, Immaturelaminin receptor, IMMP2L, INDX, INFGR1, INFGR2, INFα, IFN□□□INFγ, INS,INSR, INVS, IP-10, IP2, IPF1, IP1, IRF6, IRS1, ISCW, ITGA2, ITGA2B,ITGA6, ITGA7, ITGB2, ITGB3, ITGB4, ITIH1, ITM2B, IV, IVD, JAG1, JAK3,JBS, JBTS1, JMS, JPD, KAL1, KAL2, KALI, KLK2, KLK4, KCNA1, KCNE2, KCNE1,KCNH2, KCNJ1, KCNJ2, KCNJ1, KCNQ2, KCNQ3, KCNQ4, KCNQ1, KCS, KERA, KFM,KFS, KFSD, KHK, ki-67, KIAA0020, KIAA0205, KIAA0205/m, KIF1B, KIT,KK-LC-1, KLK3, KLKB1, KM-HN-1, KMS, KNG, KNO, K-RAS/m, KRAS2, KREV1,KRT1, KRT10, KRT12, KRT13, KRT14, KRT14L1, KRT14L2, KRT14L3, KRT16,KRT16L1, KRT16L2, KRT17, KRT18, KRT2A, KRT3, KRT4, KRT5, KRT6 A, KRT6B,KRT9, KRTHB1, KRTHB6, KRT1, KSA, KSS, KWE, KYNU, L0H19CR1, LiCAM, LAGE,LAGE-1, LALL, LAMA2, LAMA3, LAMB3, LAMB1, LAMC2, LAMP2, LAP, LCA5, LCAT,LCCS, LCCS 1, LCFS2, LCS1, LCT, LDHA, LDHB, LDHC, LDLR, LDLR/FUT, LEP,LEWISY, LGCR, LGGF-PBP, LGI1, LGMD2H, LGMD1A, LGMD1B, LHB, LHCGR, LHON,LHRH, LHX3, LIF, LIG1, LIMM, LIMP2, LIPA, LIPA, LIPB, LIPC, LIVIN,L1CAM, LMAN1, LMNA, LMX1B, LOLR, LOR, LOX, LPA, LPL, LPP, LQT4, LRP5,LRS 1, LSFC, LT-β, LTBP2, LTC4S, LYL1, XCL1, LYZ, M344, MA50, MAA,MADH4, MAFD2, MAFD1, MAGE, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2,MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGEB1, MAGE-B10, MAGE-B10,MAGE-B17, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2,MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1,MAGEL2, MGB1, MGB2, MAN2A1, MAN2B1, MANBA, MANBB, MAOA, MAOB, MAPK8IP1,MAPT, MART-1, MART-2, MART2/m, MAT1A, MBL2, MBP, MBS1, MC1R, MC2R, MC4R,MCC, MCCC2, MCCC1, MCDR1, MCF2, MCKD, MCL1, MC1R, MCOLN1, MCOP, MCOR,MCP-1, MCP-2, MCP-3, MCP-4, MCPH2, MCPH1, MCS, M-CSF, MDB, MDCR, MDM2,MDRV, MDS 1, ME1, ME1/m, ME2, ME20, ME3, MEAX, MEB, MEC CCL-28, MECP2,MEFV, MELANA, MELAS, MEN1 MSLN, MET, MF4, MG50, MG50/PXDN, MGAT2, MGAT5,MGC1 MGCR, MGCT, MGI, MGP, MHC2TA, MHS2, MHS4, MIC2, MIC5, MIDI, MIF,MIP, MIP-5/HCC-2, MITF, MJD, MKI67, MKKS, MKS1, MLH1, MLL, MLLT2, MLLT3,MLLT7, MLLT1, MLS, MLYCD, MMA1a, MMP 11, MMVP1, MN/CA IX-Antigen, MNG1,MN1, MOC31, MOCS2, MOCS1, MOG, MORC, MOS, MOV18, MPD1, MPE, MPFD, MPI,MPIF-1, MPL, MPO, MPS3C, MPZ, MRE11A, MROS, MRP1, MRP2, MRP3, MRSD,MRX14, MRX2, MRX20, MRX3, MRX40, MRXA, MRX1, MS, MS4A2, MSD, MSH2, MSH3,MSH6, MSS, MSSE, MSX2, MSX1, MTATP6, MTC03, MTCO1, MTCYB, MTHFR, MTM1,MTMR2, MTND2, MTND4, MTND5, MTND6, MTND1, MTP, MTR, MTRNR2, MTRNR1,MTRR, MTTE, MTTG, MTTI, MTTK, MTTL2, MTTL1, MTTN, MTTP, MTTS1, MUC1,MUC2, MUC4, MUC5AC, MUM-1, MUM-1/m, MUM-2, MUM-2/m, MUM-3, MUM-3/m, MUT,mutant p21 ras, MUTYH, MVK, MX2, MXI1, MY05A, MYB, MYBPC3, MYC, MYCL2,MYH6, MYH7, MYL2, MYL3, MYMY, MYO15A, MYO1G, MYO5A, MYO7A, MYOC,Myosin/m, MYP2, MYP1, NA88-A, N-acetylglucosaminyltransferase-V, NAGA,NAGLU, NAMSD, NAPB, NAT2, NAT, NBIA1, NBS1, NCAM, NCF2, NCF1, NDN, NDP,NDUFS4, NDUFS7, NDUFS8, NDUFV1, NDUFV2, NEB, NEFH, NEM1, Neo-PAP,neo-PAP/m, NEU1, NEUROD1, NF2, NF1, NFYC/m, NGEP, NHS, NKS1, NKX2E, NM,NME1, NMP22, NMTC, NODAL, NOG, NOS3, NOTCH3, NOTCH1, NP, NPC2, NPC1,NPHL2, NPHP1, NPHS2, NPHS1, NPM/ALK, NPPA, NQO1, NR2E3, NR3C1, NR3C2,NRAS, NRAS/m, NRL, NROB1, NRTN, NSE, NSX, NTRK1, NUMA1, NXF2, NY-CO1,NY-ESO1, NY-ESO-B, NY-LU-12, ALDOA, NYS2, NYS4, NY-SAR-35, NYS1, NYX,OA3, OA1, OAP, OASD, OAT, OCA1, OCA2, OCD1, OCRL, OCRL1, OCT, ODDD,ODT1, OFC1, OFD1, OGDH, OGT, OGT/m, OPA2, OPA1, OPD1, OPEM, OPG, OPN,OPN1LW, OPN1MW, OPN1SW, OPPG, OPTB1, TTD, ORM1, ORP1, OS-9, OS-9/m, OSMLIF, OTC, OTOF, OTSC1, OXCT1, OYTES1, P15, P190 MINOR BCR-ABL, P2RY12,P3, P16, P40, P4HB, P-501, P53, P53/m, P97, PABPN1, PAFAH1B1, PAFAH1P1,PAGE-4, PAGE-5, PAH, PAI-1, PAI-2, PAK3, PAP, PAPPA, PARK2, PART-1,PATE, PAX2, PAX3, PAX6, PAX7, PAX8, PAX9, PBCA, PBCRA1, PBT, PBX1,PBXP1, PC, PCBD, PCCA, PCCB, PCK2, PCK1, PCLD, PCOS1, PCSK1, PDB1, PDCN,PDE6A, PDE6B, PDEF, PDGFB, PDGFR, PDGFRL, PDHA1, PDR, PDX1, PECAM1,PEE1, PEO1, PEPD, PEX10, PEX12, PEX13, PEX3, PEX5, PEX6, PEX7, PEX1,PF4, PFBI, PFC, PFKFB1, PFKM, PGAM2, PGD, PGK1, PGK1P1, PGL2, PGR, PGS,PHA2A, PHB, PHEX, PHGDH, PHKA2, PHKA1, PHKB, PHKG2, PHP, PHYH, PI, PI3,PIGA, PIM1-KINASE, PIN1, PIP5K1B, PITX2, PITX3, PKD2, PKD3, PKD1, PKDTS,PKHD1, PKLR, PKP1, PKU1, PLA2G2A, PLA2G7, PLAT, PLEC1, PLG, PLI, PLOD,PLP1, PMEL17, PML, PML/RARα, PMM2, PMP22, PMS2, PMS1, PNKD, PNLIP, POF1,POLA, POLH, POMC, PON2, PON1, PORC, POTE, POU1F1, POU3F4, POU4F3,POU1F1, PPAC, PPARG, PPCD, PPGB, PPH1, PPKB, PPMX, PPDX, PPP1R3A,PPP2R2B, PPT1, PRAME, PRB, PRB3, PRCA1, PRCC, PRD, PRDX5/m, PRF1, PRG4,PRKAR1A, PRKCA, PRKDC, PRKWNK4, PRNP, PROC, PRODH, PROM1, PROP1, PROS1,PRST, PRP8, PRPF31, PRPF8, PRPH2, PRPS2, PRPS1, PRS, PRSS7, PRSS1,PRTN3, PRX, PSA, PSAP, PSCA, PSEN2, PSEN1, PSG1, PSGR, PSM, PSMA,PSORS1, PTC, PTCH, PTCH1, PTCH2, PTEN, PTGS1, PTH, PTHR1, PTLAH, PTOS1,PTPN12, PTPNI1, PTPRK, PTPRK/m, PTS, PUJO, PVR, PVRL1, PWCR, PXE, PXMP3,PXR1, PYGL, PYGM, QDPR, RAB27A, RAD54B, RAD54L, RAG2, RAGE, RAGE-1,RAG1, RAP1, RARA, RASA1, RBAF600/m, RB1, RBP4, RBP4, RBS, RCA1, RCAS1,RCCP2, RCD1, RCV1, RDH5, RDPA, RDS, RECQL2, RECQL3, RECQL4, REG1A,REHOBE, REN, RENBP, RENS1, RET, RFX5, RFXANK, RFXAP, RGR, RHAG,RHAMM/CD168, RHD, RHO, Rip-1, RLBP1, RLN2, RLN1, RLS, RMD1, RMRP, ROM1,ROR2, RP, RP1, RP14, RP17, RP2, RP6, RP9, RPD1, RPE65, RPGR, RPGRIP1,RP1, RP10, RPS19, RPS2, RPS4X, RPS4Y, RPS6KA3, RRAS2, RS1, RSN, RSS,RU1, RU2, RUNX2, RUNX1, RWS, RYR1, S-100, SAA1, SACS, SAG, SAGE, SALL1,SARDH, SART1, SART2, SART3, SAS, SAX1, SCA2, SCA4, SCA5, SCA7, SCA8,SCA1, SCC, SCCD, SCF, SCLC1, SCN1A, SCN1B, SCN4A, SCN5A, SCNN1A, SCNN1B,SCNN1G, SCO2, SCP1, SCZD2, SCZD3, SCZD4, SCZD6, SCZD1, SDF-1α/□□□SDHA,SDHD, SDYS, SEDL, SERPENA7, SERPINA3, SERPINA6, SERPINA1, SERPINC1,SERPIND1, SERPINE1, SERPINF2, SERPING1, SERPINI1, SFTPA1, SFTPB, SFTPC,SFTPD, SGCA, SGCB, SGCD, SGCE, SGM1, SGSH, SGY-1, SH2D1A, SHBG, SHFM2,SHFM3, SHFM1, SHH, SHOX, SI, SIAL, SIALYL LEWISX, SIASD, S11, SIM1,SIRT2/m, SIX3, SJS1, SKP2, SLC10A2, SLC12A1, SLC12A3, SLC17A5, SLC19A2,SLC22A1L, SLC22A5, SLC25A13, SLC25A15, SLC25A20, SLC25A4, SLC25A5,SLC25A6, SLC26A2, SLC26A3, SLC26A4, SLC2A1, SLC2A2, SLC2A4, SLC3A1,SLC4A1, SLC4A4, SLC5A1, SLC5A5, SLC6A2, SLC6A3, SLC6A4, SLC7A7, SLC7A9,SLC11A1, SLOS, SMA, SMAD1, SMAL, SMARCB1, SMAX2, SMCR, SMCY, SM1, SMN2,SMN1, SMPD1, SNCA, SNRPN, SOD2, SOD3, SOD1, SOS1, SOST, SOX9, SOX10,Sp17, SPANXC, SPG23, SPG3A, SPG4, SPG5A, SPG5B, SPG6, SPG7, SPINK1,SPINK5, SPPK, SPPM, SPSMA, SPTA1, SPTB, SPTLC1, SRC, SRD5A2, SRPX, SRS,SRY, ßhCG, SSTR2, SSX1, SSX2 (HOM-MEL-40/SSX2), SSX4, ST8, STAMP-1,STAR, STARP1, STATH, STEAP, STK2, STK11, STn/KLH, STO, STOM, STS, SUOX,SURF1, SURVIVIN-2B, SYCP1, SYM1, SYN1, SYNS1, SYP, SYT/SSX, SYT-SSX-1,SYT-SSX-2, TA-90, TAAL6, TACSTD1, TACSTD2, TAG72, TAF7L, TAF1, TAGE,TAG-72, TALI, TAM, TAP2, TAP1, TAPVR1, TARC, TARP, TAT, TAZ, TBP, TBX22,TBX3, TBX5, TBXA2R, TBXAS1, TCAP, TCF2, TCF1, TCIRG1, TCL2, TCL4, TCL1A,TCN2, TCOF1, TCR, TCRA, TDD, TDFA, TDRD1, TECK, TECTA, TEK, TEL/AML1,TELAB1, TEX15, TF, TFAP2B, TFE3, TFR2, TG, TGFA, TGF-□□β, TGFBI, TGFB1,TGFBR2, TGFBRE, TGFβ, TGFβRII, TGIF, TGM-4, TGM1, TH, THAS, THBD, THC,THC2, THM, THPO, THRA, THRB, TIMM8A, TIMP2, TIMP3, TIMP1, TITF1, TKCR,TKT, TLP, TLR1, TLR10, TLR2, TLR3, TLR4, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLX1, TM4SF1, TM4SF2, TMC1, TMD, TMIP, TNDM, TNF, TNFRSF11A,TNFRSF1A, TNFRSF6, TNFSF5, TNFSF6, TNFα, TNFβ, TNNI3, TNNT2, TOC, TOP2A,TOP1, TP53, TP63, TPA, TPBG, TPI, TPI/m, TPI1, TPM3, TPM1, TPMT, TPO,TPS, TPTA, TRA, TRAG3, TRAPPC2, TRC8, TREH, TRG, TRH, TRIM32, TRIM37,TRP1, TRP2, TRP-2/6b, TRP-2/INT2, Trp-p8, TRPS1, TS, TSC2, TSC3, TSC1,TSG101, TSHB, TSHR, TSP-180, TST, TTGA2B, TTN, TTPA, TTR, TU M2-PK,TULP1, TWIST, TYH, TYR, TYROBP, TYROBP, TYRP1, TYS, UBE2A, UBE3A, UBE1,UCHL1, UFS, UGT1A, ULR, UMPK, UMPS, UOX, UPA, UQCRC1, URO5, UROD, UPK1B,UROS, USH2A, USH3A, USH1A, USH1C, USP9Y, UV24, VBCH, VCF, VDI, VDR,VEGF, VEGFR-2, VEGFR-1, VEGFR-2/FLK-1, VHL, VIM, VMD2, VMD1, VMGLOM,VNEZ, VNF, VP, VRNI, VWF, VWS, WAS, WBS2, WFS2, WFS1, WHCR, WHN, WISP3,WMS, WRN, WS2A, WS2B, WSN, WSS, WT2, WT3, WT1, WTS, WWS, XAGE, XDH, XIC,XIST, XK, XM, XPA, XPC, XRCC9, XS, ZAP70, ZFHX1B, ZFX, ZFY, ZIC2, ZIC3,ZNF145, ZNF261, ZNF35, ZNF41, ZNF6, ZNF198, ZWS1, or from fragments orvariants thereof. Preferably, such fragments as well as variants exhibita sequence homology or identity of about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% withone of the proteins or peptides or protein or peptide sequences as shownor described above. In this context, the definition of fragments andvariants similarly applies as defined above for component (A) of theinventive transporter cargo conjugate molecule.

Effector molecules suitable as component (B) of the inventivetransporter cargo conjugate molecule may also be selected from proteinkinase inhibitors, particularly inhibitors of the protein kinase c-Junamino terminal kinase, i.e. a JNK inhibitor. Typically, a JNK inhibitorsuitable as component (B) of the inventive transporter cargo conjugatemolecule may be derived from a human or rat IB1 sequence, preferablyfrom an amino acid sequence as defined or encoded by any of sequencesaccording to SEQ ID NO: 137 (depicts the IB1 cDNA sequence from rat andits predicted amino acid sequence), SEQ ID NO: 138 (depicts the IB1protein sequence from rat encoded by the exon-intron boundary of therIB1 gene splice donor), SEQ ID NO: 139 (depicts the IB1 proteinsequence from Homo sapiens), or SEQ ID NO: 140 (depicts the IB1 cDNAsequence from Homo sapiens), more preferably from an amino acid sequenceas defined or encoded by any of sequences according to SEQ ID NO: 139(depicts the IB1 protein sequence from Homo sapiens), or SEQ ID NO: 140(depicts the IB1 cDNA sequence from Homo sapiens), or from any fragmentsor variants thereof. In this context, the definition of fragments andvariants similarly applies as defined above for component (A) of theinventive transporter cargo conjugate molecule.

Preferably, a JNK inhibitor sequence suitable as component (B) of theinventive transporter cargo conjugate molecule comprises a total lengthof less than 150 amino acid residues, preferably a range of 5 to 150amino acid residues, more preferably 10 to 100 amino acid residues, evenmore preferably 10 to 75 amino acid residues and most preferably a rangeof 10 to 50 amino acid residues, e.g. 10 to 30, 10 to 20, or 10 to 15amino acid residues. More preferably, such a JNK inhibitor sequence andthe above ranges may be selected from any of the herein mentioned JNKinhibitor sequence, even more preferably from an amino acid sequence asdefined according to SEQ ID NO: 139 or as encoded by SEQ ID NO: 140,even more preferably in the region between nucleotides 420 and 980 ofSEQ ID NO: 140 or amino acids 105 and 291 of SEQ ID NO: 139, and mostpreferably in the region between nucleotides 561 and 647 of SEQ ID NO:140 or amino acids 152 and 180 of SEQ ID NO: 139.

According to a particular embodiment, a JNK inhibitor sequence suitableas component (B) of the inventive transporter cargo conjugate moleculetypically binds JNK and/or inhibits the activation of at least one JNKactivated transcription factor, e.g. c-Jun or ATF2 (see e.g. SEQ ID NOs:147 and 148, respectively) or Elk1.

Likewise, a JNK inhibitor sequence suitable as component (B) of theinventive transporter cargo conjugate molecule preferably comprises orconsists of at least one amino acid sequence according to any one of SEQID NOs: 137 to 220, or a fragment, derivative or variant thereof. Morepreferably, the JNK inhibitor sequence as used herein may contain 1, 2,3, 4 or even more copies of an amino acid sequence according to SEQ IDNOs: 137 to 220, or a variant, fragment or derivative thereof. Ifpresent in more than one copy, these amino acid sequences according toSEQ ID NOs: 137 to 220, or variants, fragments, or derivatives thereofas used herein may be directly linked with each other without any linkersequence or via a linker sequence comprising 1 to 10, preferably 1 to 5amino acids. Amino acids forming the linker sequence are preferablyselected from glycine or proline as amino acid residues. Morepreferably, these amino acid sequences according to SEQ ID NOs: 137 to220, or fragments, variants or derivatives thereof, as used herein, maybe separated by each other by a hinge of two, three or more prolineresidues.

The JNK inhibitor sequence suitable as component (B) of the inventivetransporter cargo conjugate molecule may be composed of L-amino acids,D-amino acids, or a combination of both. Preferably, the JNK inhibitorsequences as used herein comprise at least 1 or even 2, preferably atleast 3, 4 or 5, more preferably at least 6, 7, 8 or 9 and even morepreferably at least 10 or more D- and/or L-amino acids, wherein the D-and/or L-amino acids may be arranged in the JNK inhibitor sequences asused herein in a blockwise, a non-blockwise or in an alternate manner.

According to one preferred embodiment the JNK inhibitor sequencesuitable as component (B) of the inventive transporter cargo conjugatemolecule may be exclusively composed of L-amino acids. The JNK inhibitorsequences as used herein may then comprise or consist of at least onenative JNK inhibitor sequence” according to SEQ ID NO: 141 or 143. Inthis context, the term “native” or “native JNK inhibitor sequence(s)” isreferred to non-altered JNK inhibitor sequences according to any of SEQID NOs: 141 or 143, as used herein, entirely composed of L-amino acids.

Accordingly, the JNK inhibitor sequence suitable as component (B) of theinventive transporter cargo conjugate molecule may comprise or consistof at least one (native) amino acid sequence NH₂-X_(n) ^(b)-X_(n)^(a)-RPTTLXLXXXXXXXQD-X_(n) ^(b)-COOH (L-IB generic (s))

SEQ ID NO: 143

and/or the JNK binding domain (JBDs) of IB1 XRPTTLXLXXXXXXXQDS/TX (L-IB(generic))

SEQ ID NO: 151

. In this context, each X typically represents an amino acid residue,preferably selected from any (native) amino acid residue. X_(n) ^(a)typically represents one amino acid residue, preferably selected fromany amino acid residue except serine or threonine, wherein n (the numberof repetitions of X) is 0 or 1. Furthermore, each X_(n) ^(b) may beselected from any amino acid residue, wherein n (the number ofrepetitions of X) is 0-5, 5-10, 10-15, 15-20, 20-30 or more, providedthat if n (the number of repetitions of X) is 0 for X_(n) ^(a), X_(n)^(b) does preferably not comprise a serine or threonine at itsC-terminus, in order to avoid a serine or threonine at this position.Preferably, X_(n) ^(b) represents a contiguous stretch of peptideresidues derived from SEQ ID NOs: 141 or 143. X_(n) ^(a) and X_(n) ^(b)may represent either D or L amino acids. Additionally, the JNK inhibitorsequence as used herein may comprise or consist of at least one (native)amino acid sequence selected from the group comprising the JNK bindingdomain of IB1 DTYRPKRPTTLNLFPQVPRSQDT (L-IB1)

SEQ ID NO: 149

. More preferably, the JNK inhibitor sequence as used herein further maycomprise or consist of at least one (native) amino acid sequenceNH₂—RPKRPTTLNLFPQVPRSQD-COOH (L-IB1(s))

SEQ ID NO: 141

. Furthermore, the JNK inhibitor sequence as used herein may comprise orconsist of at least one (native) amino acid sequence selected from thegroup comprising the JNK binding domain of IB1 L-IB1(s1)(NH₂-TLNLFPQVPRSQD-COOH, SEQ ID NO: 153); L-IB1(s2)(NH₂-TTLNLFPQVPRSQ-COOH, SEQ ID NO: 154); L-IB1(s3)(NH₂—PTTLNLFPQVPRS-COOH, SEQ ID NO: 155); L-IB1(s4)(NH₂—RPTTLNLFPQVPR-COOH, SEQ ID NO: 156); L-IB1(s5)(NH₂—KRPTTLNLFPQVP-COOH, SEQ ID NO: 157); L-IB1(s6)(NH₂—PKRPTTLNLFPQV-COOH, SEQ ID NO: 158); L-IB1(s7)(NH₂—RPKRPTTLNLFPQ-COOH, SEQ ID NO: 159); L-IB1(s8)(NH₂-LNLFPQVPRSQD-COOH, SEQ ID NO: 160); L-IB1(s9)(NH₂-TLNLFPQVPRSQ-COOH, SEQ ID NO: 161); L-IB1(s10)(NH₂-TTLNLFPQVPRS-COOH, SEQ ID NO: 162); L-IB1(s11)(NH₂—PTTLNLFPQVPR-COOH, SEQ ID NO: 163); L-IB1(s12)(NH₂—RPTTLNLFPQVP-COOH, SEQ ID NO: 164); L-IB1(s13)(NH₂—KRPTTLNLFPQV-COOH, SEQ ID NO: 165); L-IB1(s14)(NH₂—PKRPTTLNLFPQ-COOH, SEQ ID NO: 166); L-IB1(s1.5)(NH₂—RPKRPTTLNLFP-COOH, SEQ ID NO: 167); L-IB1(s16)(NH₂—NLFPQVPRSQD-COOH, SEQ ID NO: 168); L-IB1(s17)(NH₂-LNLFPQVPRSQ-COOH, SEQ ID NO: 169); L-IB1(s18)(NH₂-TLNLFPQVPRS-COOH, SEQ ID NO: 170); L-IB1(s19)(NH₂-TTLNLFPQVPR-COOH, SEQ ID NO: 171); L-IB1(s20)(NH₂—PTTLNLFPQVP-COOH, SEQ ID NO: 172); L-IB1(s21)(NH₂—RPTTLNLFPQV-COOH, SEQ ID NO: 173); L-IB1(s22)(NH₂—KRPTTLNLFPQ-COOH, SEQ ID NO: 174); L-IB1(s23)(NH₂—PKRPTTLNLFP-COOH, SEQ ID NO: 175); L-IB1(s24)(NH₂—RPKRPTTLNLF-COOH, SEQ ID NO: 176); L-IB1(s25) (NH₂-LFPQVPRSQD-COOH,SEQ ID NO: 177); L-IB1(s26) (NH₂—NLFPQVPRSQ-COOH, SEQ ID NO: 178);L-IB1(s27) (NH₂-LNLFPQVPRS-COOH, SEQ ID NO: 179); L-IB1(s28)(NH₂-TLNLFPQVPR-COOH, SEQ ID NO: 180); L-IB1(s29) (NH₂-TTLNLFPQVP-COOH,SEQ ID NO: 181); L-IB1(s30) (NH₂—PTTLNLFPQV-COOH, SEQ ID NO: 182);L-IB1(s31) (NH₂—RPTTLNLFPQ-COOH, SEQ ID NO: 183); L-IB1(s32)(NH₂—KRPTTLNLFP-COOH, SEQ ID NO: 184); L-IB1(s33) (NH₂—PKRPTTLNLF-COOH,SEQ ID NO: 185); and L-IB1(s34) (NH₂—RPKRPTTLNL-COOH, SEQ ID NO: 186).

Additionally, the JNK inhibitor sequence suitable as component (B) ofthe inventive transporter cargo conjugate molecule may comprise orconsist of at least one (native) amino acid sequence selected from thegroup comprising the (long) JNK binding domain (JBDs) of IBPGTGCGDTYRPKRPTTLNLFPQVPRSQDT (IB1-long)

SEQ ID NO: 145

, the (long) JNK binding domain of IB2 IPSPSVEEPHKHRPTTLRLTTLGAQDS(IB2-long)

SEQ ID NO: 146

, the JNK binding domain of c-Jun GAYGYSNPKILKQSMTLNLADPVGNLKPH (c-Jun)

SEQ ID NO: 147

, the JNK binding domain of ATF2 TNEDHLAVHKHKHEMTLKFGPARNDSVIV (ATF2)

SEQ ID NO: 148

). In this context, an alignment revealed a partially conserved 8 aminoacid sequence and a further comparison of the JBDs of IB1 and IB2revealed two blocks of seven and three amino acids that are highlyconserved between the two sequences.

According to another preferred embodiment the JNK inhibitor sequencesuitable as component (B) of the inventive transporter cargo conjugatemolecule may be composed in part or exclusively of D-amino acids asdefined above. More preferably, these JNK inhibitor sequences composedof D-amino acids are non-native D retro-inverso sequences of the above(native) JNK inhibitor sequences. The term “retro-inverso sequences”refers to an isomer of a linear peptide sequence in which the directionof the sequence is reversed and the chirality of each amino acid residueis inverted (see e.g. Jameson et al., Nature, 368, 744-746 (1994); Bradyet al., Nature, 368, 692-693 (1994)). The advantage of combiningD-enantiomers and reverse synthesis is that the positions of carbonyland amino groups in each amide bond are exchanged, while the position ofthe side-chain groups at each alpha carbon is preserved. Unlessspecifically stated otherwise, it is presumed that any given L-aminoacid sequence or peptide as used according to the present invention maybe converted into an D retro-inverso sequence or peptide by synthesizinga reverse of the sequence or peptide for the corresponding nativeL-amino acid sequence or peptide.

Accordingly, the JNK inhibitor sequence suitable as component (B) of theinventive transporter cargo conjugate molecule may comprise or consistof at least one D retro-inverso sequence according to the amino acidsequence NH₂-X_(n) ^(b)-DQXXXXXXXLXLTTPR-X_(n) ^(a)-X_(n) ^(b)-COOH(D-IB1 generic (s))

SEQ ID NO: 144

and/or XS/TDQXXXXXXXLXLTTPRX (D-IB (generic))

SEQ ID NO: 152

. As used in this context, X, X_(n) ^(a) and X_(n) ^(b) are as definedabove (preferably, representing D amino acids), wherein X_(n) ^(b)preferably represents a contiguous stretch of residues derived from SEQID NO: 142 or 144. Additionally, the JNK inhibitor sequences as usedherein may comprise or consist of at least one D retro-inverso sequenceaccording to the amino acid sequence comprising the JNK binding domain(JBDs) of IB1 TDQSRPVQPFLNLTTPRKPRYTD (D-IB1)

SEQ ID NO: 150

. More preferably, the JNK inhibitor sequences as used herein maycomprise or consist of at least one D retro-inverso sequence accordingto the amino acid sequence NH₂-DQSRPVQPFLNLTTPRKPR-COOH (D-IB1(s))

SEQ ID NO: 142

. Furthermore, the JNK inhibitor sequences as used herein may compriseor consist of at least one D retro-inverso sequence according to theamino acid sequence comprising the JNK binding domain (JBDs) of IB1D-IB1(s1) (NH₂-QPFLNLTTPRKPR-COOH, SEQ ID NO: 187); D-IB1(s2)(NH₂-VQPFLNLTTPRKP-COOH, SEQ ID NO: 188); D-IB1(s3)(NH₂—PVQPFLNLTTPRK-COOH, SEQ ID NO: 189); D-IB1(s4)(NH₂—RPVQPFLNLTTPR-COOH, SEQ ID NO: 190); D-IB1(s5)(NH₂—SRPVQPFLNLTTP-COOH, SEQ ID NO: 191); D-IB1(s6)(NH₂-QSRPVQPFLNLTT-COOH, SEQ ID NO: 192); D-IB1(s7)(NH₂-DQSRPVQPFLNLT-COOH, SEQ ID NO: 193); D-IB1(s8)(NH₂—PFLNLTTPRKPR-COOH, SEQ ID NO: 194); D-IB1(s9)(NH₂-QPFLNLTTPRKP-COOH, SEQ ID NO: 195); D-IB1(s10)(NH₂—VQPFLNLTTPRK-COOH, SEQ ID NO: 196); D-IB1(s11)(NH₂—PVQPFLNLTTPR-COOH, SEQ ID NO: 197); D-IB1(s12)(NH₂—RPVQPFLNLTTP-COOH, SEQ ID NO: 198); D-IB1(s13)(NH₂—SRPVQPFLNLTT-COOH, SEQ ID NO: 199); D-IB1(s14)(NH₂-QSRPVQPFLNLT-COOH, SEQ ID NO: 200); D-IB1(s15)(NH₂-DQSRPVQPFLNL-COOH, SEQ ID NO: 201); D-M1(s16)(NH₂—FLNLTTPRKPR-COOH, SEQ ID NO: 202); D-IB1(s17)(NH₂—PFLNLTTPRKP-COOH, SEQ ID NO: 203); D-IB1(s18)(NH₂-QPFLNLTTPRK-COOH, SEQ ID NO: 204); D-IB1(s19)(NH₂—VQPFLNLTTPR-COOH, SEQ ID NO: 205); D-IB1(s20)(NH₂—PVQPFLNLTTP-COOH, SEQ ID NO: 206); D-IB1(s21)(NH₂—RPVQPFLNLTT-COOH, SEQ ID NO: 207); D-IB1(s22)(NH₂—SRPVQPFLNLT-COOH, SEQ ID NO: 208); D-IB1(s23)(NH₂-QSRPVQPFLNL-COOH, SEQ ID NO: 209); D-IB1(s24)(NH₂-DQSRPVQPFLN-COOH, SEQ ID NO: 210); D-IB1(s25) (NH₂-DQSRPVQPFL-COOH,SEQ ID NO: 211); D-IB1(s26) (NH₂-QSRPVQPFLN-COOH, SEQ ID NO: 212);D-IB1(s27) (NH₂—SRPVQPFLNL-COOH, SEQ ID NO: 213); D-IB1(s28)(NH₂—RPVQPFLNLT-COOH, SEQ ID NO: 214); D-IB1(s29) (NH₂—PVQPFLNLTT-COOH,SEQ ID NO: 215); D-IB1(s30) (NH₂—VQPFLNLTTP-COOH, SEQ ID NO: 216);D-IB1(s31) (NH₂-QPFLNLTTPR-COOH, SEQ ID NO: 217); D-IB1(s32)(NH₂—PFLNLTTPRK-COOH, SEQ ID NO: 218); D-IB1(s33) (NH₂—FLNLTTPRKP-COOH,SEQ ID NO: 219); and D-IB1(s34) (NH₂-LNLTTPRKPR-COOH, SEQ ID NO: 220).

Exemplary JNK inhibitor sequence suitable as component (B) of theinventive transporter cargo conjugate molecule and are presented inTable 4 (SEQ ID NO:s 141 to 220). The table presents the name of the JNKinhibitor sequences as used herein, as well as their sequence identifiernumber, their length, and amino acid sequence. Furthermore, Table 4shows IB1 derived sequences as well as their generic formulas, e.g. forSEQ ID NO's: 141 and 142 and SEQ ID NO's: 143 and 144, respectively.Table 4 furthermore discloses L-IB1 sequences according to SEQ ID NOs:153 to 186 and D-IB1 sequences SEQ ID NOs: 187 to 220.

TABLE 4 SEQUENCE/ SEQ PEPTIDE ID NAME NO AA SEQUENCE L-IB1(s) 141 19RPKRPTTLNLFPQVPRSQD (NH₂-RPKRPTTLNLFPQVPRSQD-COOH) D-IB1(s) 142 19DQSRPVQPFLNLTTPRKPR (NH₂-DQSRPVQPFLNLTTPRKPR-COOH) L-IB (generic) (s)143 19 NH₂-X_(n) ^(b)-X_(n) ^(a)-RPTTLXLXXXXXXXQD-X_(n) ^(b)-COOHD-IB (generic) (s) 144 19 NH₂-X_(n) ^(b)-DQXXXXXXXLXLTTPR-X_(n)^(a)-X_(n) ^(b)-COOH IB1-long 145 29 PGTGCGDTYRPKRPTTLNLFPQVPRSQDT(NH₂-PGTGCGDTYRPKRPTTLNLFPQVPRSQDT- COOH) IB2-long 146 27IPSPSVEEPHKHRPTTLRLTTLGAQDS (NH₂-IPSPSVEEPHKHRPTTLRLTTLGAQDS-COOH) c-Jun147 29 GAYGYSNPKILKQSMTLNLADPVGNLKPH (NH₂-GAYGYSNPKILKQSMTLNLADPVGNLKPH-COOH) ATF2 148 29 TNEDHLAVHKHKHEMTLKFGPARNDSVIV(NH₂-TNEDHLAVHKHKHEMTLKFGPARNDSVIV- COOH) L-IB1 149 23DTYRPKRPTTLNLFPQVPRSQDT (NH₂-DTYRPKRPTTLNLFPQVPRSQDT-COOH) D-IB1 150 23TDQSRPVQPFLNLTTPRKPRYTD (NH₂-TDQSRPVQPFLNLTTPRKPRYTD-COOH)L-IB (generic) 151 19 XRPTTLXLXXXXXXXQDS/TX(NH₂-XRPTTLXLXXXXXXXQDS/TX-COOH) D-IB (generic) 152 19XS/TDQXXXXXXXLXLTTPRX (NH₂-XS/TDQXXXXXXXLXLTTPRX-COOH) L-IB1(s1) 153 13TLNLFPQVPRSQD (NH₂-TLNLFPQVPRSQD-COOH) L-IB1(s2) 154 13 TTLNLFPQVPRSQ(NH₂-TTLNLFPQVPRSQ-COOH) L-IB1(s3) 155 13 PTTLNLFPQVPRS(NH₂-PTTLNLFPQVPRS-COOH) L-IB1(s4) 156 13 RPTTLNLFPQVPR(NH₂-RPTTLNLFPQVPR-COOH) L-IB1(s5) 157 13 KRPTTLNLFPQVP(NH₂-KRPTTLNLFPQVP-COOH) L-IB1(s6) 158 13 PKRPTTLNLFPQV(NH₂-PKRPTTLNLFPQV-COOH) L-IB1(s7) 159 13 RPKRPTTLNLFPQ(NH₂-RPKRPTTLNLFPQ-COOH) L-IB1(s8) 160 12 LNLFPQVPRSQD(NH₂-LNLFPQVPRSQD-COOH) L-IB1(s9) 161 12 TLNLFPQVPRSQ(NH₂-TLNLFPQVPRSQ-COOH) L-IB1(s10) 162 12 TTLNLFPQVPRS(NH₂-TTLNLFPQVPRS-COOH) L-IB1(s11) 163 12 PTTLNLFPQVPR(NH₂-PTTLNLFPQVPR-COOH) L-IB1(s12) 164 12 RPTTLNLFPQVP(NH₂-RPTTLNLFPQVP-COOH) L-IB1(s13) 165 12 KRPTTLNLFPQV(NH₂-KRPTTLNLFPQV-COOH) L-IB1(s14) 166 12 PKRPTTLNLFPQ(NH₂-PKRPTTLNLFPQ-COOH) L-IB1(s15) 167 12 RPKRPTTLNLFP(NH₂-RPKRPTTLNLFP-COOH) L-IB1(s16) 168 11 NLFPQVPRSQD(NH₂-NLFPQVPRSQD-COOH) L-IB1(s17) 169 11 LNLFPQVPRSQ(NH₂-LNLFPQVPRSQ-COOH) L-IB1(s18) 170 11 TLNLFPQVPRS(NH₂-TLNLFPQVPRS-COOH) L-IB1(s19) 171 11 TTLNLFPQVPR(NH₂-TTLNLFPQVPR-COOH) L-IB1(s20) 172 11 PTTLNLFPQVP(NH₂-PTTLNLFPQVP-COOH) L-IB1(s21) 173 11 RPTTLNLFPQV(NH₂-RPTTLNLFPQV-COOH) L-IB1(s22) 174 11 KRPTTLNLFPQ(NH₂-KRPTTLNLFPQ-COOH) L-IB1(s23) 175 11 PKRPTTLNLFP(NH₂-PKRPTTLNLFP-COOH) L-IB1(s24) 176 11 RPKRPTTLNLF(NH₂-RPKRPTTLNLF-COOH) L-IB1(s25) 177 10 LFPQVPRSQD(NH₂-LFPQVPRSQD-COOH) L-IB1(s26) 178 10 NLFPQVPRSQ (NH₂-NLFPQVPRSQ-COOH)L-IB1(s27) 179 10 LNLFPQVPRS (NH₂-LNLFPQVPRS-COOH) L-IB1(s28) 180 10TLNLFPQVPR (NH₂-TLNLFPQVPR-COOH) L-IB1(s29) 181 10 TTLNLFPQVP(NH₂-TTLNLFPQVP-COOH) L-IB1(s30) 182 10 PTTLNLFPQV (NH₂-PTTLNLFPQV-COOH)L-IB1(s31) 183 10 RPTTLNLFPQ (NH₂-RPTTLNLFPQ-COOH) L-IB1(s32) 184 10KRPTTLNLFP (NH₂-KRPTTLNLFP-COOH) L-IB1(s33) 185 10 PKRPTTLNLF(NH₂-PKRPTTLNLF-COOH) L-IB1(s34) 186 10 RPKRPTTLNL (NH₂-RPKRPTTLNL-COOH)D-IB1(s1) 187 13 QPFLNLTTPRKPR (NH₂-QPFLNLTTPRKPR-COOH) D-IB1(s2) l88 13VQPFLNLTTPRKP (NH₂-VQPFLNLTTPRKP-COOH) D-IB1(s3) 189 13 PVQPFLNLTTPRK(NH₂-PVQPFLNLTTPRK-COOH) D-IB1(s4) 190 13 RPVQPFLNLTTPR(NH₂-RPVQPFLNLTTPR-COOH) D-IB1(s5) 191 13 SRPVQPFLNLTTP(NH₂-SRPVQPFLNLTTP-COOH) D-IB1(s6) 192 13 QSRPVQPFLNLTT(NH₂-QSRPVQPFLNLTT-COOH) D-IB1(s7) 193 13 DQSRPVQPFLNLT(NH₂-DQSRPVQPFLNLT-COOH) D-IB1(s8) 194 12 PFLNLTTPRKPR(NH₂-PFLNLTTPRKPR-COOH) D-IB1(s9) 195 12 QPFLNLTTPRKP(NH₂-QPFLNLTTPRKP-COOH) D-IB1(s10) 196 12 VQPFLNLTTPRK(NH₂-VQPFLNLTTPRK-COOH) D-IB1(s11) 197 12 PVQPFLNLTTPR(NH₂-PVQPFLNLTTPR-COOH) D-IB1(s12) 198 12 RPVQPFLNLTTP(NH₂-RPVQPFLNLTTP-COOH) D-IB1(s13) 199 12 SRPVQPFLNLTT(NH₂-SRPVQPFLNLTT-COOH) D-IB1(s14) 200 12 QSRPVQPFLNLT(NH₂-QSRPVQPFLNLT-COOH) D-IB1(s15) 201 12 DQSRPVQPFLNL(NH₂-DQSRPVQPFLNL-COOH) D-IB1(s16) 202 11 FLNLTTPRKPR(NH₂-FLNLTTPRKPR-COOH) D-IB1(s17) 203 11 PFLNLTTPRKP(NH₂-PFLNLTTPRKP-COOH) D-IB1(s18) 204 11 QPFLNLTTPRK(NH₂-QPFLNLTTPRK-COOH) D-IB1(s19) 205 11 VQPFLNLTTPR(NH₂-VQPFLNLTTPR-COOH) D-IB1(s20) 206 11 PVQPFLNLTTP(NH₂-PVQPFLNLTTP-COOH) D-IB1(s21) 207 11 RPVQPFLNLTT(NH₂-RPVQPFLNLTT-COOH) D-IB1(s22) 208 11 SRPVQPFLNLT(NH₂-SRPVQPFLNLT-COOH) D-IB1(s23) 209 11 QSRPVQPFLNL(NH₂-QSRPVQPFLNL-COOH) D-IB1(s24) 210 11 DQSRPVQPFLN(NH₂-DQSRPVQPFLN-COOH) D-IB1(s25) 211 10 DQSRPVQPFL(NH₂-DQSRPVQPFL-COOH) D-IB1(s26) 212 10 QSRPVQPFLN (NH₂-QSRPVQPFLN-COOH)D-IB1(s27) 213 10 SRPVQPFLNL (NH₂-SRPVQPFLNL-COOH) D-IB1(s28) 214 10RPVQPFLNLT (NH₂-RPVQPFLNLT-COOH) D-IB1(s29) 215 10 PVQPFLNLTT(NH₂-PVQPFLNLTT-COOH) D-IB1(s30) 216 10 VQPFLNLTTP (NH₂-VQPFLNLTTP-COOH)D-IB1(s31) 217 10 QPFLNLTTPR (NH₂-QPFLNLTTPR-COOH) D-IB1(s32) 218 10PFLNLTTPRK (NH₂-PFLNLTTPRK-COOH) D-IB1(s33) 219 10 FLNLTTPRKP(NH₂-FLNLTTPRKP-COOH) D-IB1(s34) 220 10 LNLTTPRKPR (NH₂-LNLTTPRKPR-COOH)

The JNK inhibitor sequences suitable as component (B) of the inventivetransporter cargo conjugate molecule may furthermore comprises orconsists of at least one variant, fragment and/or derivative of theabove defined native or non-native amino acid sequences according to SEQID NOs: 141 to 220. Preferably, these variants, fragments and/orderivatives retain biological activity of the above disclosed native ornon-native JNK inhibitor sequences as used herein, particularly ofnative or non-native amino acid sequences according to SEQ ID NOs: 141to 220, i.e. binding JNK and/or inhibiting the activation of at leastone JNK activated transcription factor, e.g. c-Jun, ATF2 or Elk 1.Functionality may be tested by various tests, e.g. binding tests of thepeptide to its target molecule or by biophysical methods, e.g.spectroscopy, computer modeling, structural analysis, etc. Particularly,an JNK inhibitor sequence or variants, fragments and/or derivativesthereof as defined above may be analyzed by hydrophilicity analysis (seee.g. Hopp and Woods, 1981. Proc Natl Acad Sci USA 78: 3824-3828) thatcan be utilized to identify the hydrophobic and hydrophilic regions ofthe peptides, thus aiding in the design of substrates for experimentalmanipulation, such as in binding experiments, or for antibody synthesis.Secondary structural analysis may also be performed to identify regionsof an JNK inhibitor sequence or of variants, fragments and/orderivatives thereof as used herein that assume specific structuralmotifs (see e.g. Chou and Fasman, 1974, Biochem 13: 222-223).Manipulation, translation, secondary structure prediction,hydrophilicity and hydrophobicity profiles, open reading frameprediction and plotting, and determination of sequence homologies can beaccomplished using computer software programs available in the art.Other methods of structural analysis include, e.g. X-ray crystallography(see e.g. Engstrom, 1974. Biochem Exp Biol 11: 7-13), mass spectroscopyand gas chromatography (see e.g. METHODS IN PROTEIN SCIENCE, 1997, J.Wiley and Sons, New York, N.Y.) and computer modeling (see e.g.Fletterick and Zoller, eds., 1986. Computer Graphics and MolecularModeling, In: CURRENT COMMUNICATIONS IN MOLECULAR BIOLOGY, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.) may also be employed.

Accordingly, the JNK inhibitor sequence suitable as component (B) of theinventive transporter cargo conjugate molecule may comprise or consistof at least one variant of (native or non-native) amino acid sequencesaccording to SEQ ID NOs: 141 to 220. In the context of the presentinvention, a “variant of a (native or non-native) amino acid sequenceaccording to SEQ ID NOs: 141 to 220” is preferably a sequence derivedfrom any of the sequences according to SEQ ID NOs: 141 to 220, whereinthe variant comprises amino acid alterations of the amino acid sequencesaccording to SEQ ID NOs: 141 to 220. Such alterations typically comprise1 to 20, preferably 1 to 10 and more preferably 1 to 5 substitutions,additions and/or deletions of amino acids according to SEQ ID NOs: 141to 220, wherein the variant exhibits a sequence identity with any of thesequences according to SEQ ID NOs: 141 to 220 of at least about 30%,50%, 70%, 80%, 90%, 95%, 98% or even 99%. If variants of (native ornon-native) amino acid sequences according to SEQ ID NOs: 141 to 220 asdefined above and used herein are obtained by substitution of specificamino acids, such substitutions preferably comprise conservative aminoacid substitutions as already defined above.

Effector molecules suitable as component (B) of the inventivetransporter cargo conjugate molecule may furthermore be selected fromantigens or antigenic fragments, preferably protein and peptideantigens, e.g. tumor antigens or antigenic fragments thereof, allergyantigens or antigenic fragments thereof, auto-immune self-antigens orantigenic fragments thereof, pathogenic antigens or antigenic fragmentsthereof, and antigens or antigenic fragments thereof from viruses,preferably from cytomegalovirus (CMV), orthopox variola virus, orthopoxalastrim virus, parapox ovis virus, molluscum contagiosum virus, herpessimplex virus 1, herpes simplex virus 2, herpes B virus, varicellazoster virus, pseudorabies virus, human cytomegaly virus, human herpesvirus 6, human herpes virus 7, Epstein-Barr virus, human herpes virus 8,hepatitis B virus, chikungunya virus, O'nyong'nyong virus, rubivirus,hepatitis C virus, GB virus C, West Nile virus, dengue virus, yellowfever virus, louping ill virus, St. Louis encephalitis virus, Japan Bencephalitis virus, Powassan virus, FSME virus, SARS, SARS-associatedcorona virus, human corona virus 229E, human corona virus Oc43,Torovirus, human T cell lymphotropic virus type I, human T celllymphotropic virus type II, HIV (AIDS), i.e. human immunodeficiencyvirus type 1 or human immunodeficiency virus type 2, influenza virus,Lassa virus, lymphocytic choriomeningitis virus, Tacaribe virus, Juninvirus, Machupo virus, Borna disease virus, Bunyamwera virus, Californiaencephalitis virus, Rift Valley fever virus, sand fly fever virus,Toscana virus, Crimean-Congo haemorrhagic fever virus, Hazara virus,Khasan virus, Hantaan virus, Seoul virus, Prospect Hill virus, Puumalavirus, Dobrava Belgrade virus, Tula virus, sin nombre virus, LakeVictoria Marburg virus, Zaire Ebola virus, Sudan Ebola virus, IvoryCoast Ebola virus, influenza virus A, influenza virus B, influenzaviruses C, parainfluenza virus, malaria virus, Marburg virus, measlesvirus, mumps virus, respiratory syncytial virus, human metapneumovirus,vesicular stomatitis Indiana virus, rabies virus, Mokola virus,Duvenhage virus, European bat lyssavirus 1+2, Australian bat lyssavirus,adenoviruses A-F, human papilloma viruses, condyloma virus 6, condylomavirus 11, polyoma viruses, adeno-associated virus 2, rotaviruses,orbiviruses, Varicella including Varizella zoster, etc., or antigens orantigenic fragments from leishmania, typanosomes, amibes, bacteria,etc., or may be selected from epitopes or from variants of the aboveantigens or antigenic fragments. Preferably, fragments as well asvariants of antigens as defined above exhibit a sequence homology oridentity of about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, or about 90% with one of the antigens orantigen sequences as shown or described above. In this context, thedefinition of fragments and variants similarly applies as defined abovefor component (A) of the inventive transporter cargo conjugate molecule.Furthermore, epitopes (also called “antigen determinants”) of antigensor antigenic fragments as defined above are encompassed, Epitopes in thecontext of the present invention are typically fragments located on theouter surface of (native) protein or peptide antigens as defined herein,preferably having 5 to 15 amino acids, more preferably having 5 to 12amino acids, even more preferably having 6 to 9 amino acids, which maybe recognized by antibodies, i.e. in their native form.

Furthermore, effector molecules suitable as component (B) of theinventive transporter cargo conjugate molecule may be selected fromantibodies. According to the present invention, such an antibody may beselected from any antibody, e.g. any recombinantly produced or naturallyoccurring antibodies, known in the art, in particular antibodiessuitable for therapeutic, diagnostic or scientific purposes, orantibodies which have been identified in relation to specific cancerdiseases. Herein, the term “antibody” is used in its broadest sense andspecifically covers monoclonal and polyclonal antibodies (includingagonist, antagonist, and blocking or neutralizing antibodies) andantibody species with polyepitopic specificity. According to theinvention, “antibody” typically comprises any antibody known in the art(e.g. IgM, IgD, IgG, IgA and IgE antibodies), such as naturallyoccurring antibodies, antibodies generated by immunization in a hostorganism, antibodies which were isolated and identified from naturallyoccurring antibodies or antibodies generated by immunization in a hostorganism and recombinantly produced by biomolecular methods known in theart, as well as chimeric antibodies, human antibodies, humanizedantibodies, bispecific antibodies, intrabodies, i.e. antibodiesexpressed in cells and optionally localized in specific cellcompartments, and fragments and variants of the aforementionedantibodies. In general, an antibody consists of a light chain and aheavy chain both having variable and constant domains. The light chainconsists of an N-terminal variable domain, V_(L), and a C-terminalconstant domain, C_(L). In contrast, the heavy chain of the IgGantibody, for example, is comprised of an N-terminal variable domain,V_(H), and three constant domains, C_(H)1, C_(H)2 and C_(H)3. Antibodiesin this context also comprise fragments and variants of antibodies asdescribed above, e.g. an F_(ab) fragment, an F_(c) fragment, etc.Preferably, such fragments as well as variants exhibit a sequencehomology or identity of about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, or about 90% with one of theantibodies as described above. In this context, the definition offragments and variants similarly applies as defined above for component(A) of the inventive transporter cargo conjugate molecule.

Additionally, effector molecules suitable as component (B) of theinventive transporter cargo conjugate molecule may be selected fromapoptotic factors or apoptosis related proteins including AIF, Apaf e.g.Apaf-1, Apaf-2, Apaf-3, oder APO-2 (L), APO-3 (L), Apopain, Bad, Bak,Bax, Bcl-2, Bcl-x_(L), Bcl-x_(s), bik, Bok, CAD, Calpain, Caspase e.g.Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6,Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, ced-3, ced-9,c-Jun, c-Myc, crm A, cytochrom C, CdR1, DcR1, DD, DED, DISC,DNA-PK_(cs), DR3, DR4, DR5, FADD/MORT-1, FAK, Fas (Fas-ligand CD95/fas(receptor)), FLICE/MACH, FLIP, fodrin, fos, G-Actin, Gas-2, gelsolin,granzyme A/B, ICAD, ICE, JNK, lamin A/B, MAP, Max, MCL-1, Mdm-2, MEKK-1,MORT-1, Myd88, NEDD, NF-_(kappa)B, NuMa, p38, p53, PAK-2, PARP,perforin, PITSLRE, PKCdelta, pRb, presenilin, prICE, RAIDD, Ras, RIP,sphingomyelinase, thymidinkinase from herpes simplex, TRADD, TRAF2,TRAIL-R1, TRAIL-R2, TRAIL-R3, transglutaminase, etc., or from fragmentsor variants thereof, or from components of the wnt-signalling pathway,such as β-catenine, or the ICF-family, pololike kinases, CiP2A, PP2A,etc., or from fragments or variants thereof. Preferably, such fragmentsas well as variants exhibit a sequence homology or identity of about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, or about 90% with one of the sequences as shown or describedabove. In this context, the definition of fragments and variantssimilarly applies as defined above for component (A) of the inventivetransporter cargo conjugate molecule.

Effector molecules suitable as component (B) of the inventivetransporter cargo conjugate molecule may furthermore be selected from atleast one or more partial or full-length BH3-domain and/or at least onepartial or full-length BH3-only protein. In this context, BH3-onlyproteins are preferably defined as members of the Bcl-2 familyrepresenting regulators of apoptosis by interacting with other membersof Bcl-2 family. In the context of the present invention component (B)of the inventive transporter cargo conjugate molecule may thus beselected from an amino acid sequence comprising at least one or morepartial or full-length BH3-domain sequence(s) of a BH3-only protein or apartial or full-length BH3-only protein (defined as a subclass of theBcl-2 family proteins), which is (are) capable of inducing apoptosis byeither interacting with at least one Bcl-2 family protein or byactivating or sensitising at least one pro-apoptotic member of the Bcl-2family. Their functional activity can be tested by suitable assaymethods, e.g. by binding assays or by assaying its pro-apoptoticactivity by apoptosis assays. Preferably, an amino acid sequence used ascomponent (B) of the inventive transporter cargo conjugate molecule maycomprise or consist of at least one partial or full-length BH3-domainsequence and/or at least one partial or full-length BH3-only proteinsequence selected from the group consisting of Bid, Bad, Noxa, Puma,Bim, Bik, Bmf, DP5/Hrk and Bok. Alternatively, an amino acid sequenceused as component (B) of the inventive transporter cargo conjugatemolecule may comprise or consist of a combination of at least onepartial or full-length BH3-domain sequence and/or at least one partialor full-length BH3-only protein sequence, the combinations preferablyselected from the group consisting of e.g. Bid and Bad, Bim and Bad, Bikand Bad, Puma and Bad, Noxa and Bad, Bmf and Bad, DP5/Hrk and Bad, Bokand Bad, Bik and Bim, Bik and Bid, Bik and Puma, Bik and Noxa, Bik andBmf, Bik and DP5/Hrk, Bik and Bok, Bid and Puma, Bid and Noxa, Bid andBim, Bid and Bmf, Bid and DP5/Hrk, Bid and Bok, Bim and Noxa, Bim andPuma, Bim and Bmf, Bim and DP5/Hrk, Bim and Bok, Puma and Noxa, Puma andBmf, Puma and DP5/Hrk, Puma and Bok, Noxa and Bmf, Noxa and DP5/Hrk andNoxa and Bok. The (full-length or partial) BH3-sequences or BH3-onlyprotein sequences defined above may be selected from e.g. any mammalianBH3-only protein, in particular from the human isoforms. Accordingly,component (B) of the inventive transporter cargo conjugate molecule maycomprise or consist of at least one partial or full-length BH3-domainsequence and/or at least one BH3-only protein sequence as defined by anyof SEQ ID NOs: 221 to 237 (see Table 5). Preferably, an amino acidsequence used as component (B) of the inventive transporter cargoconjugate molecule may further comprise or consist of at least onefragment or variant of at least one partial or full-length BH3-domainsequence and/or at least one BH3-only protein sequence as defined by anyof SEQ ID NOs: 221 to 237. Such fragments as well as variants preferablyhave a sequence length of less than 50, preferably of less than 40 andeven more preferably of less than 30 amino acids, or exhibit a sequencehomology or identity of about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, or about 90% with one of thesequences described above or as shown in any of SEQ ID NOs: 221 to 237.In this context, the definition of fragments and variants similarlyapplies as defined above for component (A) of the inventive transportercargo conjugate molecule. Furthermore, fragments or variants of thenative sequences typically comprise a BH3-domain sequence or at leastpartially comprise a BH3-domain sequence (at least 7 amino acids of theBH3-domain sequence).

TABLE 5 SEQUENCE/ SEQ PEPTIDE NAME ID NO AA SEQUENCE Bid (human) 221 241MCSCACVMMA RWAARGRAGW (transcript RSTVRILSPL GHCEPGVSRS variant 1)CRAAQAMDCE VNNGSSLRDE CITNLLVFGF LQSCSDNSFR RELDALGHEL PVLAPQWEGYDELQTDGNRS SHSRLGRIEA DSESQEDIIR NIARHLAQVG DSMDRSIPPG LVNGLALQLRNTSRSEEDRN RDLATALEQL LQAYPRDMEK EKTMLVLALL LAKKVASHTP SLLRDVFHTTVNFINQNLRT YVRSLARNGM D Bad (human) 222 168 MFQIPEFEPS EQEDSSSAERGLGPSPAGDG PSGSGKHHRQ APGLLWDASH QQEQPTSSSH HGGAGAVEIR SRHSSYPAGTEDDEGMGEEP SPFRGRSRSA PPNLWAAQRY GRELRRMSDE FVDSFKKGLP RPKSAGTATQMRQSSSWTRV FQSWWDRNLG RGSSAPSQ Noxa1 (human) 223 483MASLGDLVRA WHLGAQAVDR GDWARALHLF SGVPAPPARL CFNAGCVHLL AGDPEAALRAFDQAVTKDTC MAVGFFQRGV ANFQLARFQE ALSDFWLALE QLRGHAAIDY TQLGLRFKLQAWEVLHNVAS AQCQLGLWTE AASSLREAMS KWPEGSLNGL DSALDQVQRR GSLPPRQVPRGEVFRPHRWH LKHLEPVDFL GKAKVVASAI PDDQGWGVRP QQPQGPGANH DARSLIMDSPRAGTHQGPLD AETEVGADRC TSTAYQEQRP QVEQVGKQAP LSPGLPAMGG PGPGPCEDPAGAGGAGAGGS EPLVTVTVQC AFTVALRARR GADLSSLRAL LGQALPHQAQ LGQLSYLAPGEDGHWVPIPE EESLQRAWQD AAACPRGLQL QCRGAGGRPV LYQVVAQHSY SAQGPFDLGFRQGDTVDVLC EEPDVPLAVD QAWLEGHCDG RIGIFPKCFV VPAGPRMSGA PGRLPRSQQC DQPPuma (human) 224 193 MARARQEGSS PEPVEGLARD GPRPFPLGRL VPSAVSCGLCEPGLAAAPAA PTLLPAAYLC APTAPPAVTA ALGCSRWPGG PRSRPRGPRP DGPQPSLSLAEQHLESPVPS APGALAGGPT QAAPGVRGEE EQWAREIGAQ LRRMADDLNA QYERRRQEEQQRHRPSPWRV LYNLIMGLLP LPRGHRAPEM EPN Bim (human) 225 198MAKQPSDVSS ECDREGRQLQ (transcript PAERPPQLRP GAPTSLQTEP variant 1)QGNPEGNHGG EGDSCPHGSP QGPLAPPASP GPFATRSPLF IFMRRSSLLS RSSSGYFSFDTDRSPAPMSC DKSTQTPSPP CQAFNHYLSA MASMRQAEPA DMRPEIWIAQ ELRRIGDEFNAYYARRVFLN NYQAAEPHPR MVILRLLRYI VRLVWRMH Bik (human) 226 160MSEVRPLSRD ILMETLLYEQ LLEPPTMEVL GMTDSEEDLD PMEDFDSLEC MEGSDALALRLACIGDEMDV SLRAPRLAQL SEVAMHSLGL AFIYDQTEDI RDVLRSFMDG FTTLKENIMRFWRSPNPGSW VSCEQVLLAL LLLLALLLPL LSGGLHLLLK BH3-domain of 227 18ALALRLACIG DEMDVSLR Bik (Bik BH3) BH3-domain of 228 18RYGREIRRMS DEFVDSFK Bad (Bad BH3) BH3-domain of 229 18NIARHLAQVG DSMDRSIP Bid (Bid BH3) BH3-domain of 230 18QIARKLQCIA DQFHRLHV Bmf (Bmf BH3) BH3-domain of 231 18LTAARLKAIG DELHQRTM DP5/Hrk (DP5Hrk BH3) BH3-domain of 232 18WIAQELRRIG DEFNAYYA Bim (Bim BH3) BH3-domain of 233 18ECATQLRRFG DKLNFRQK Noxa (Noxa BH3) BH3-domain of 234 18EIGAQLRRMA DDLNAQYE PUMA (PUMA BH3) BH3-domain of 235 18KLSECLKRIG DELDSNME Bax (Bax BH3) BH3-domain of 236 18QVGRQLAIIG DDINRRYD Bak (Bak BH3) BH3-domain of 237 18EVCTVLLRLC DELEQIRP Bok (Bok BH3)

The proteins or peptide sequences as described above, e.g. oftherapeutically active proteins, antigens, antibodies, apoptoticfactors, proteases implicated in pathological states, preferablypeptidic protease inhibitors, BH3 domains, etc., which are used aseffector molecules for component (B) of the inventive transporter cargoconjugate molecule, may be provided in as a protein or peptide sequenceeither in the native form composed of L-amino acids or in theretro-inverso D-form ((entirely) composed of D amino acids, which meansthat these sequences have to be inverted by reverting the termini:native C-terminus is the N-terminus of the inverted form and the nativeN-terminus is the C-Terminus of the inverted form). Alternatively, theseproteins or peptide sequences as described above, may provide theirprotein or peptide sequence as a mixture of L-amino acids and D-aminoacids.

Effector molecules suitable as component (B) of the inventivetransporter cargo conjugate molecule may additionally be selected fromnucleic acids, preferably from nucleic acids encoding the above definedproteins or peptides, such as therapeutically active proteins andpeptides, antigens, antibodies, apoptotic factors, proteases implicatedin pathological states, preferably peptidic protease inhibitors,BH3-domains or partial or full-length BH3-only proteins or theirvariants of fragments. In this context, nucleic acids preferablycomprise single stranded, double stranded or partially double strandednucleic acids, preferably selected from genomic DNA, cDNA, RNA, siRNA,antisense DNA, antisense RNA, ribozyme, complimentary RNA/DNA sequenceswith or without expression elements, a mini-gene, gene fragments,regulatory elements, promoters, and combinations thereof.

As a further particular example, effector molecules suitable ascomponent (B) of the inventive transporter cargo conjugate molecule maybe selected from siRNAs. In this context, an siRNAs is of interestparticularly in connection with the phenomenon of RNA interference.Attention was drawn to the phenomenon of RNA interference in the courseof immunological research. In recent years, a RNA-based defencemechanism has been discovered, which occurs both in the kingdom of thefungi and in the plant and animal kingdom and acts as an “immune systemof the genome”. The system was originally described in various speciesindependently of one another, first in C. elegans, before it waspossible to identify the underlying mechanisms of the processes as beingidentical: RNA-mediated virus resistance in plants, PTGS(posttranscriptional gene silencing) in plants, and RNA interference ineukaryotes are accordingly based on a common procedure. The in vitrotechnique of RNA interference (RNAi) is based on double-stranded RNAmolecules (dsRNA), which trigger the sequence-specific suppression ofgene expression (Zamore (2001) Nat. Struct. Biol. 9: 746-750; Sharp(2001) Genes Dev. 5:485-490: Hannon (2002) Nature 41: 244-251). In thetransfection of mammalian cells with long dsRNA, the activation ofprotein kinase R and RnaseL brings about unspecific effects, such as,for example, an interferon response (Stark et al. (1998) Annu. Rev.Biochem. 67: 227-264; He and Katze (2002) Viral Immunol. 15: 95-119).These unspecific effects are avoided when shorter, for example 21-to23-mer, so-called siRNA (small interfering RNA), is used, becauseunspecific effects are not triggered by siRNA that is shorter than 30 bp(Elbashir et al. (2001) Nature 411: 494-498). Recently, dsRNA moleculeshave also been used in vivo (McCaffrey et al. (2002), Nature 418: 38-39;Xia et al. (2002), Nature Biotech. 20: 1006-1010; Brummelkamp et al.(2002), Cancer Cell 2: 243-247). Thus, an siRNA used as an effectormolecule suitable as component (B) of the inventive transporter cargoconjugate molecule typically comprises a (single- or) double stranded,preferably a double-stranded, RNA sequence with about 8 to 30nucleotides, preferably 17 to 25 nucleotides, even more preferably from20 to 25 and most preferably from 21 to 23 nucleotides. In principle,all the sections having a length of from 17 to 29, preferably from 19 to25, most preferably from 21 to 23 base pairs that occur in the codingregion of a protein (sequence) as mentioned above, can serve as targetsequence for a siRNA. Equally, siRNAs can also be directed againstnucleotide sequences of a protein (sequence) described hereinbefore thatdo not lie in the coding region, in particular in the 5′ non-codingregion of the RNA, for example, therefore, against non-coding regions ofthe RNA having a regulatory function. The target sequence of the siRNAcan therefore lie in the translated and/or untranslated region of theRNA and/or in the region of the control elements. The target sequence ofa siRNA can also lie in the overlapping region of untranslated andtranslated sequence; in particular, the target sequence can comprise atleast one nucleotide upstream of the start triplet of the coding region.

As another particular example, effector molecules suitable as component(B) of the inventive transporter cargo conjugate molecule may beselected from antisense RNA. In this context, an antisense RNA ispreferably a (single-stranded) RNA molecule transcribed on the basis ofthe coding, rather than the template, strand of (genomic) DNA, so thatit is complementary to the sense (messenger) RNA. An antisense RNAsuitable as component (B) of the inventive transporter cargo conjugatemolecule typically forms a duplex between the sense and antisense RNAmolecules and is thus capable to block translation of the correspondingmRNA. An antisense RNA as used herein can be directed against anyportion of an mRNA sequence, e.g. derived from genomic DNA and/or whichmay encode any protein, e.g. a protein peptide as defined herein such astherapeutically active proteins and peptides, antigens, antibodies,apoptotic factors, proteases implicated in pathological states,preferably peptidic protease inhibitors, BH3-domains or partial orfull-length BH3-only proteins or their variants of fragments asdescribed hereinbefore, if thereby translation of the encoded protein orpeptide is reduced/suppressed. Accordingly, the target sequence of theantisense RNA on the targeted mRNA (or the targeted (genomic) DNA) maybe located in the translated and/or untranslated region of the mRNA (orthe targeted (genomic) DNA), e.g. in the region of the control elements,in particular in the 5′ non-coding region of the mRNA (or the targeted(genomic) DNA) exerting a regulatory function. The target sequence of anantisense RNA on the targeted mRNA (or the targeted (genomic) DNA) mayalso be constructed such that the antisense RNA binds to the mRNA (orthe targeted (genomic) DNA) by covering with its sequence a region whichis partially complementary to the untranslated and to translated(coding) sequence of the targeted mRNA (or the targeted (genomic) DNA);in particular, the antisense RNA may be complementary to the target mRNA(or the targeted (genomic) DNA) sequence by at least one nucleotideupstream of the start triplet of the coding region of the targeted mRNA.Preferably, the antisense RNA as used herein comprises a length of about5 to about 5000, of about 500 to about 5000, and, more preferably, ofabout 1000 to about 5000 or, alternatively, of about 5 to about 1000,about 5 to about 500, about 5 to about 250, of about 5 to about 100, ofabout 5 to about 50 or of about 5 to about 30 nucleotides, or,alternatively, and even more preferably a length of about 20 to about100, of about 20 to about 80, or of about 20 to about 60 nucleotides.

As a further particular example, effector molecules suitable ascomponent (B) of the inventive transporter cargo conjugate molecule mayadditionally be selected from a cytotoxic or anti-tumor drug, suitableas a chemotherapy drug. In general, chemotherapy drugs suitable forcomponent (B) of the inventive transporter cargo conjugate molecule canbe divided into three maincategories based on their mechanism of action.They may (a) stop the synthesis of preDNA molecule building blocks:These agents work in a number of different ways. DNA building blocks arefolic acid, heterocyclic bases, and nucleotides, which are madenaturally within cells. All of these agents work to block some step inthe formation of nucleotides or deoxyribonucleotides (necessary formaking DNA). When these steps are blocked, the nucleotides, which arethe building blocks of DNA and RNA, cannot be synthesized. Thus thecells cannot replicate because they cannot make DNA without thenucleotides. Examples of drugs in this class include methotrexate(Abitrexate®), fluorouracil (Adrucil®), hydroxyurea (Hydrea®), andmercaptopurine (Purinethol®), thioguanine, tocoferol, or, moregenerally, also any nucleotide analogue, e.g. 2′-deoxycytidineanalogues. Alternatively, chemotherapy drugs may (b) directly damage theDNA in the nucleus of the cell. These agents chemically damage DNA andRNA. They disrupt replication of the DNA and either totally haltreplication or cause the manufacture of nonsense DNA or RNA (i.e. thenew DNA or RNA does not code for anything useful). Examples of drugs inthis class include cisplatin (Platinol®) and antibiotics-daunorubicin(Cerubidine®), doxorubicin (Adriamycin®) belonging to the class ofanthracycline antitumor agents (the members of which may be used ascomponent (B) of the inventive transporter cargo conjugate molecule),and etoposide (VePesid®) or any intercalator. Finally, chemotherapydrugs may (c) effect the synthesis or breakdown of the mitotic spindles:Mitotic spindles serve as molecular railroads with “North and SouthPoles” in the cell when a cell starts to divide itself into two newcells. These spindles are very important because they help to split thenewly copied DNA such that a copy goes to each of the two new cellsduring cell division. These drugs disrupt the formation of thesespindles and therefore interrupt cell division. Examples of drugs inthis class of mitotic disrupters include: Vinblastine (Velban®),Vincristine (Oncovin®) and Paclitaxel (Taxol®). Component (B) of theinventive transporter cargo conjugate molecule may act according to oneof the above modes of action. In other terms, each of the classes ofanti-tumor drugs, i.e. alkylating agents, nitrosoureas, antimetabolites,plant alkaloids, antitumor antibiotics, and steroid hormones may be usedas component (B) of the inventive transporter cargo conjugate molecule.To describe these drug classes in more detail it is emphasized that eachanti cancer drug may also be categorized according to its effect on thecell cycle and cell chemistry as disclosed above. Alkylating agents killcells by directly attacking DNA. Alkylating agents may be used in thetreatment of chronic leukemias, Hodgkin's disease, lymphomas, andcertain carcinomas of the lung, breast, prostate and ovary.Cyclophosphamide is an example of a commonly used alkylating agent.Nitrosoureas act similarly to akylating agents and also inhibit changesnecessary for DNA repair. These agents cross the blood-brain barrier andare therefore used to treat brain tumors, lymphomas, multiple myeloma,and malignant melanoma. Carmustine and lomustine are the major drugs inthis category. Antimetabolites are that drugs block cell growth byinterfering with certain activities, usually DNA synthesis. Onceingested into the cell they halt normal development and reproduction.All drugs in this category affect the cell during the “S” phase of thecell cycle. Antimetabolites may be used in the treatment of acute andchronic leukemias, choriocarcinoma, and some tumors of thegastrointestinal tract, breast and ovary. Examples of commonly usedantimetabolites are 6-mercaptopurine and 5-fluorouracil (5FU). Antitumorantibiotics are a diverse group of compounds. In general, they act bybinding with DNA and preventing RNA synthesis. These agents are widelyused in the treatment of a variety of cancers. The most commonly useddrugs in this group are doxorubicin (Adriamycin), mitomycin-C, andbleomycin. Plant (vinca)alkaloids are anti-tumor agents derived fromplants. These drugs act specifically by blocking cell division duringmitosis. They are commonly used in the treatment of acute lymphoblasticleukemia, Hodgkin's and non-Hodgkin's lymphomas, neuroblastomas, Wilms'tumor, and cancers of the lung, breast and testes. Vincristine andvinblastine are commonly used agents in this group. Steroid hormones areuseful in treating some types of tumors. This class includesadrenocorticosteroids, estrogens, antiestrogens, progesterones, andandrogens. Although their specific mechanism of action is not clear,steroid hormones modify the growth of certain hormone-dependent cancers.Tamoxifen is an example, which is used for estrogen dependent breastcancer. All of the above-mentioned tumor species may be treated by theinventive transporter cargo conjugate molecules comprising as component(B) any of the above antitumor agents.

One group of cytotoxic or anti-tumor drugs, which may be used aseffector molecules for component (B) of the inventive transporter cargoconjugate molecule is preferably selected from alkylating drugs,antimetabolica, cytostatics or drugs related to hormone treatment. Inthis context, it it is preferred to select as cytotoxic or anti-tumordrugs compounds of metal, in particular platin (derivative) and taxolclasses. In particular, the drug moiety is selected from the group ofdrugs consisting of, for example, cisplatin, transplatin, satraplatin,oxaliplatin, carboplatin, nedaplatin, chlorambucil, cyclophosphamide,mephalan, azathioprin, fluorouracil, (6)-mercaptopurine, methrexate,nandrolone, aminogluthemide, medroxyprogesteron, megestrolacetate,procarbazin, docetaxel, paclitaxel, irinotecan, epipodophyllotoxin,podophyllotoxin, vincristine, vinblastine, docetaxel, daunomycin,daunorubicin, doxorubicin, mitoxantrone, topotecan, bleomycin,gemcitabine, fludarabine, navelbine and 5-FUDR. Particularly preferredis the class of metal containing anticancer drugs, e.g. the class ofplatinum compounds.

Further cytotoxic or anti-tumor drugs, which may be used as effectormolecules for component (B) of the inventive transporter cargo conjugatemolecule are (identified by their generic name) Alitretinoin,Altretamine, Azathioprine, Bicalutamide, Busulfan, Capecitabine,Cyclophosphamide, Exemestane, Letrozole, Finasteride, Megestrol Acetate,Triptorelin, Temozolomide, Mifepristone, Tretinoin, Oral, Tamoxifen,Teniposide, Imatinib (Gleevec®), Gefitinib (IRESSA®), Peplomycin sulfateor the class of camptothecins.

Another group of cytotoxic or anti-tumor drugs, which may be used aseffector molecules for component (B) of the inventive transporter cargoconjugate molecule are indolocarbazole compounds, e.g. staurosporin (andits analogues) and rebeccamycin. It is to be mentioned that compoundsbelonging to the class of anilinoquinazolines (e.g. gefitinib) are alsoparticularly preferred as component (B).

A further group of cytotoxic or anti-tumor drugs, which may be used aseffector molecules for component (B) of the inventive transporter cargoconjugate molecule may additionally be selected from inhibitors oftopoisomerases, such as irinotecan, or mitotic kinesins or DHFR.

Additionally, cytotoxic or anti-tumor drugs, which may be used aseffector molecules for component (B) of the inventive transporter cargoconjugate molecule can be selected from factors inhibiting orstimulating cell proliferation (PDGF), intracellular pathways, e.g. theRAS/RAF signaling pathway, such as a member of the RAF/MEK/ERK signalingpathway (e.g. RAF-1) or mitogen-activated protein kinase pathway, CMGCkinase family (containing CDK (cyclin dependent-kinases), MAPK, GSK3,CLK), Ser/Thr kinases that belong to the AGC kinase family containingPKA, PKG, PKC kinase families, receptor tyrosine kinases involved e.g.in neovascularization and tumor progression, including vascularendothelial growth factor receptor (VEGFR)-2, VEGFR-3, platelet-derivedgrowth factor receptor ß, Flt-3, the endothelin (ET) system, thatincludes ET-1, ET-2, ET-3, and the ET_(A) receptor (ET_(A)R) andET_(B)R, and c-KIT, which are targeted by e.g. inhibiting theirfunction, and members of the IGF-family, such as IGF-1, IGF-2, IGF-1R,IGF2R, etc.

Another group of cytotoxic or anti-tumor drugs, which may be used aseffector molecules for component (B) of the inventive transporter cargoconjugate molecule may may be selected from inhibitors that target tumorcell proliferation and tumor angiogenesis. Particularly preferred inthis context are small molecule antitumor kinase inhibitors directedtoward targets on malignant cells and/or vascular cells haveantiangiogenic activity. Kinase inhibitors such as those directed towardEGFR, Her2/neu, BCR-ABL, c-KIT, PKC, Raf and PI3, are antiangiogenic byvirtue of blocking secretion of angiogenic factors by affected malignantcells. Kinase inhibitors such as those directed toward VEGFR2, VEGFR1,PDGFR, PKC, Raf and PI3, are antiangiogenic by effects on vascularcells. Examples of synthetic inhibitors of cyclin dependent kinases(CDKIs) are e.g. olomoucine, flavopiridol, butyrolactone and theirderivatives and thus constrain tumor cell proliferation. On the otherhand, antitumor compounds suitable as component (B) of the inventivetransporter cargo conjugate molecule may be selected from activators ofapoptosis programs in cancer cells (e.g. staurosporine) or bydownregulating antiapoptotic proteins, e.g. Bcl-2.

It is common to all of the above compounds that they have to cross thecell membrane in order to act as anticancer drugs. By coupling compoundsbelonging to each of these classes (compounds directly damaging the DNAin the nucleus of the cell, effecting the synthesis or breakdown of themitotic spindles or stopping the synthesis of pre-DNA molecule buildingblocks) as component (B) to component (A) to form the inventivetransporter cargo conjugate molecule, the entry of the anticancercompounds into the cell is enhanced and/or their solubility is enhanced,thereby increasing the efficacy of these therapeutic compounds. In turn,increased cell take-up and, preferably, better solubility of thesecompounds in the aqueous environment (e.g. the cytosol) allows to lowerthe dosage of the therapeutic anti-cancer compound.

Additionally, component (B) of the inventive transporter cargo conjugatemolecule may also comprise small organic compounds or drug molecules,such as protease inhibitors which inhibit proteases, in particularproteases which are involved in the infection cycle of infectiousagents, e.g. viral, bacterial or protozoological proteases. In apreferred embodiment, these protease inhibitors (organic compounds ordrug molecules) as part of an inventive conjugate molecule may serve totreat viral, bacterial infections or protozoological infections, e.g.malaria. In particular, virus infections may be treated by proteaseinhibitors, e.g. retroviral diseases. The use of conjugate moleculescomprising protease inhibitors are strongly preferred for the treatmentof HIV infections. The protease inhibitors to be used for coupling tocarrier sequence as disclosed herein may be selected from a groupcontaining the 640385, abacavir sulfate, AG1776, amprenavir (141W94 orVX-478), atazanavir (BMS-232632), Cathepsin S protease inhibitor, D1927,D9120, efavirenz, emtricitabine, enfuvirtide (T-20), fosamprenavir(GW-433908 or VX-175), GS 9005, GW640385 (VX-385), HCV proteaseinhibitor, indinavir (MK-639), L-756, 423, levoprin-ZG, lopinavir(ABT-378), lopinavir/ritonavir (LPV ABT-378/r), MK-944A, mozenavir(DMP450), nelfinavir (AG-1343), nevirapine, P-1946, PL-100, prinomastat,ritonavir (ABT-538), RO033-4649, TMC114, saquinavir (Ro-31-8959),tenofovir disoproxil fumarate, tipranavir (PNU-140690), TLK 19781,TMC-114, Vertex 385, VX-950.

Finally, effector molecules suitable as component (B) of the inventivetransporter cargo conjugate molecule may additionally be selected as aseparate component from a label as defined above for the inventivetransporter cargo conjugate molecule. Such an inventive transportercargo conjugate molecule is particularly suitable for in vitro or invivo assays. In this context, labels may comprise radioactive labels,i.e. radioactive phosphorylation or a radioactive label with sulphur,hydrogen, carbon, nitrogen, etc.; colored dyes (e.g. digoxygenin, etc.);fluorescent groups (e.g. fluorescein, rhodamine, flourochrome proteinsas defined below, etc.); chemoluminescent groups; or combination ofthese labels. Preferably, flourochrome proteins comprise anyfluorochrome protein, which can be activated such as to emit afluorescence signal. More preferably, the fluorochrome protein isselected from any fluorescent protein, e.g. from a group comprising theGreen Fluorescent Protein (GFP), derivatives of the Green FluorescentProtein (GFP), e.g. EGFP, AcGFP, TurboGFP, Emerald, Azami Green, thephoto activatable-GFP (PA-GFP), or Blue Fluorescent Protein (BFP)including EBFP, Sapphire, T-Sapphire, or Cyan Fluorescent Proteins (CFP)including the enhanced cyan fluorescent protein (ECFP), mCFP, Cerulan,CyPet, or Yellow Fluorescent Proteins (YFP), including Topaz, Venus,mCitrine, Ypet, PhiYFP, mBanana, the yellow shifted green fluorescentprotein (Yellow GFP), the enhanced yellow fluorescent protein (EYFP), orOrange and Red Flourescent Proteins (RFP) including Kusibara Orange,mOrange, dTomato-Tandem, DsRed-Monomer, mTangerine, mStrawberry,monomeric red fluorescent protein (mRFP1) (also designated herein asmRFP), mCherry, mRaspberry, HcRed-Tandem, mPlum, as well as opticalhighlighters selected from PA-GFP, CoralHue Dronpa (G), PS-CFP (C),PS-CFP (G), mEosFP (G), mEosFP (G), or other monomeric fluorescentproteins such as or the kindling fluorescent protein (KFP1), aequorin,the autofluorescent proteins (AFPs), or the fluorescent proteins JRed,TurboGFP, PhiYFP and PhiYFP-m, tHc-Red (HcRed-Tandem), PS-CFP2 andKFP-Red (as available from EVRΩGEN, see also www.evrogen.com), or othersuitable fluorescent proteins.

The inventive transporter cargo conjugate molecule comprising components(A) and (B) may furthermore comprise at least one optional additionalcomponent (C), (D) and/or (E), etc., preferably different to component(B). This at least one optional additional portion may award additionalfunctions to the inventive fusion protein and may be selectedindependent from other components (B), (C), (D) and/or (E).

For example, the at least one optional additional component (C), (D)and/or (E), etc., of the inventive transporter cargo conjugate moleculemay be any of the effector molecules as described for component (B)above. Preferably, the at least one optional additional component (C),(D) and/or (E), etc., of the inventive transporter cargo conjugatemolecule is not identical to the specifically selected component (B) ofthe inventive transporter cargo conjugate molecule, i.e., the at leastone optional additional component (C), (D) and/or (E), etc., preferablymay be selected independent from each other from different effectormolecules or their fragments or variants as described above. The atleast one optional additional component (C), (D) and/or (E), etc., ofthe inventive transporter cargo conjugate molecule can furthermore be anamino acid, oligopeptide or polypeptide or a (small) organo-chemicalcompound and can be linked to the inventive transporter cargo conjugatemolecule at a suitable position, for example, the N-terminus, theC-terminus of the inventive transporter cargo conjugate molecule or maybe internally coupled to amino acids, e.g. amino acid side chains, or tonucleic acids or any suitable position of a component (B) (or (A)). Theat least one optional additional component (C), (D) and/or (E), etc. ofthe inventive transporter cargo conjugate molecule can also be a portion(e.g., HA, HSV-Tag, His6-Tag, FLAG-Tag), which may render the inventivetransporter cargo conjugate molecule amenable to purification and/orisolation. If desired, the component needed for purification can then beremoved from the other components of the inventive transporter cargoconjugate molecule (e.g., by proteolytic cleavage or other methods knownin the art) at the end of the production process.

Furthermore, the at least one optional additional component (C), (D)and/or (E), etc. of the inventive transporter cargo conjugate moleculemay be a signal sequence or localisation sequence, which efficientlydirects the inventive transporter cargo conjugate molecule to aparticular intracellular target localization, preferably without loss ofthe enhanced cell permeability properties of the inventive transportercargo conjugate molecule. Typically, such a signal sequence orlocalisation sequence directs the inventive transporter cargo conjugatemolecule to specific cell compartments, e.g., endoplasmic reticulum,mitochondrion, gloom apparatus, lysosomal vesicles, etc. Exemplarysignal sequences or localisation sequences include, without beinglimited thereto, localisation sequences for the endoplasmic reticulum,such as KDEL (SEQ ID NO: 238), DDEL (SEQ ID NO: 239), DEEL (SEQ ID NO:240), QEDL (SEQ ID NO: 241), RDEL (SEQ ID NO: 242), sequences for thelocalisation into the nucleus, such as PKKKRKV (SEQ ID NO: 243), PQKKIKS(SEQ ID NO: 244), QPKKP (SEQ ID NO: 245), RKKR (SEQ ID NO: 246),sequences for the localisation for the nuclear region, such asRKKRRQRRRAHQ (SEQ ID NO: 247), RQARRNRRRRWRERQR (SEQ ID NO: 248),MPLTRRRPAASQALAPPTP (SEQ ID NO: 249), sequences for the localisationinto the endodomal compartiment, such as MDDQRDLISNNEQLP (SEQ ID NO:250), etc.

Similarly, the at least one optional additional component (C), (D)and/or (E), etc. of the inventive transporter cargo conjugate moleculemay be a signal sequence or localisation sequence, which efficientlydirects the inventive transporter cargo conjugate molecule to aparticular cell type, preferably without loss of the enhanced cellpermeability properties of the inventive transporter cargo conjugatemolecule.

The inventive transporter cargo conjugate molecule may furthermorecomprise at least one modification, preferably at its termini, either atthe C- or the N-terminus or both. The C-terminus may preferably bemodified by an amide modification, whereas the N-terminus may bemodified by any suitable NH₂-protection group, such as e.g. acylation,or any further modification as already indicated above for L-aminoacids. Such modifications also includes introduction of labels asdefined above for inventive transporter molecules according to generalformula (I) above.

Finally, the components (B), (C), (D), and/or (E) of the inventivetransporter cargo conjugate molecule as described above as well aslinkers, which may be optionally used for interlinking these components,may comprise or consist of protein or peptide sequences. Such protein orpeptide sequences may be composed of L-amino acids, D-amino acids, or acombination of both, preferably as described above for the inventivenovel novel transporter construct according to generic formula (I) oraccording to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If)above. Such D- and/or L-amino acids may be arranged in the components(B), (C), (D), and/or (E) in a blockwise, a non-blockwise or in analternate manner. Alternatively, a pattern as described above for theinventive novel novel transporter construct according to generic formula(I) or according to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or(If) above may be repeated fort these components. In other words,generic formula (I) (SEQ ID NO: 1) D_(l)LLL_(x)D_(m)(LLL_(y)D_(m))_(a)as defined above, may be applied, wherein the number of repeats asdefined by a is not limited to a range of 0-3 but may be applied to theentire molecule, i.e. a will be 1 to 500, 1 to 250, 1 to 100, 1 to 50, 1to 20, 1 to 10, 1 to 5 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., and ispreferably determined by the length of the protein or peptide sequences,which is to be covered.

The components (A), (B) of the inventive transporter cargo conjugatemolecule and, if present, further optional components (C), (D) and/or(E), etc. are typically coupled with each other, via covalent bonds orvia electrostatic bonding (e.g. poly-lysine), preferably via covalentbonds. In this context the term “covalent bond” relates to a stablechemical link between two atoms produced by sharing one or more pairs ofelectrons. Preferably, all components (A) and (B) of the inventivetransporter cargo conjugate molecule and, if present, further optionalcomponents (C), (D) and/or (E), etc. may be coupled as to form a linearmolecule or a non-linear (branched) molecule, preferably a linearmolecule. In a linear molecule, all the above components (A) and (B)and, if present, optional components (C), (D) and/or (E), etc. arelinked to each other via their terminal ends of in a linear form withoutleading to branched transporter cargo conjugate molecule. In anon-linear (branched) molecule all the above components (A) and (B) and,if present, optional components (C), (D) and/or (E), etc. are linked toeach other via their terminal ends of in a form which heads to abranched transporter cargo conjugate molecule, e.g. having an Y-shapedform, etc.

As component (A) of the inventive transporter cargo conjugate moleculeis per definition a peptide sequence consisting of D- and L-amino acids,the (covalent) attachment of further components (B) and, if present, ofoptional components (C), (D) and/or (E), may, of course, depend on thetype and nature of the components to be attached, i.e. as to whether thesingle components are proteins or peptides, nucleic acids, (small)organic compounds, etc.

The order, in which component (B) of the inventive transporter cargoconjugate molecule and, if present, further optional components (C), (D)and/or (E), are linked with component (A) and each other to form apreferably linear molecule, typically may comprise any order.Accordingly, any of components (A), (B), and if present, (C), (D) and/or(E) may be attached with each other. However, component (A) ispreferably attached at the terminal ends of the inventive transportercargo conjugate molecule. If any of components (B), and if present,components (C), (D) and/or (E), is a protein or a peptide sequence,component (A) is preferably contained at the C-terminal end of theinventive transporter cargo conjugate molecule, e.g., at the C-terminalend of component (B) as defined above or, if present, of components (C),(D) and/or (E), when occurring as a peptide or a protein. Such aposition of component (A) in the inventive transporter cargo conjugatemolecule prevents the cargo peptide or protein sequence of components(B), (C), (D) and/or (E) to be degraded prior to its/their transport tothe desired target site, e.g. the cell, the nucleus, etc., by apeptidase, particularly a carboxy peptidase such as carboxyterminalpeptidase N. Alternatively, if there are aminoterminal peptidases in thecell systems used, the component (A) may be located at the aminoterminalend of the inventive transporter cargo conjugate molecule.

If the further component (B) and/or, if present, any of the optionalcomponent (C), (D) and/or (E), is a peptide or protein sequence, thelink between these protein or peptide components of the inventivetransporter cargo conjugate molecule is typically a peptide bond. Such apeptide bond may be formed using a chemical synthesis involving bothcomponents (an N-terminal end of one component and the C-terminal end ofthe other component) to be linked, or may be formed directly via aprotein synthesis of the entire peptide sequence of both components,wherein both (protein or peptide) components are preferably synthesizedin one step. Such protein synthesis methods include e.g., without beinglimited thereto, liquid phase peptide synthesis methods or solid peptidesynthesis methods, e.g. solid peptide synthesis methods according toMerrifield, t-Boc solid-phase peptide synthesis, Fmoc solid-phasepeptide synthesis, BOP(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate) based solid-phase peptide synthesis, etc.

Furthermore, component (A) and component (B) of the inventivetransporter cargo conjugate molecule can be coupled via a linker ordirectly (without linker) by e.g. an amide bridge, if the components tobe linked have reactive amino or carboxy groups. Alternatively, ester orether linkages are preferred.

If present, further components (C), (D) and/or (E), etc., as mentionedabove, can be coupled in an analogous manner to component (A) and/orcomponent (B) or, optionally, with each other to then be linked as onesingle moiety to either component (A) or component (B). Linker sequencescan also be used to fuse the components of inventive transporter cargoconjugate molecule with at least one other component (see below). Themode of coupling further component(s) to the either component (A) orcomponent (B) of the inventive transporter cargo conjugate molecule willdepend on its chemical character. If additional components (C), (D),(E), etc., belong to the class of peptidic sequences, they willpreferably linked to the inventive transporter cargo conjugate moleculeto either terminus of component (A) or, alternatively, be linked viacomponent (A)'s L- or D amino acid side chains, e.g. by a disulfidebridge. Further components of other chemical nature may be likewiseattached to component (A) (terminal groups or chemically active sidechain groups) or component (B). The linkage via a side chain willpreferably be based on side chain amino, thiol or hydroxyl groups, e.g.via an amide or ester or ether linkage. It has to be noted that,according to the invention, all amino acids (of any of component (A),and, if built of amino acids, components (C), (D), (E) etc.) arepreferably D-enantiomeric amino acids, which reflect its eventuallynaturally occurring analogue by being linked in retro-inverso order.Nevertheless, components (C), (D), (E) etc., if composed of amino acids,may also be composed of L-amino acids (in their naturally occurringsequence order) or built of a combination of D and L amino acids.

If peptidic linker sequences are used to fuse component (A) and (B) orto fuse another component, e.g. (C) to component (A) and/or (B), thelinker sequences preferably form a flexible sequence of 2 to 10residues, more preferably 1 to 5 residues. In a preferred embodiment thelinker sequence contains at least 20%, more preferably at least 40% andeven more preferably at least 50% Gly or β-alanine residues, e.g.GlyGlyGlyGlyGly (SEQ ID NO: 255), GlyGlyGlyGly (SEQ ID NO: 256),GlyGlyGly, CysGlyGly or GlyGlyCys, etc. Appropriate linker sequences canbe easily selected and prepared by a person skilled in the art. They maybe composed of D and/or L amino acids.

Peptide linker sequences may also be introduced between a component (A)and a component (B), and/or further components (C), (D) and/or (E), ofthe inventive transporter cargo conjugate molecule, wherein anamino-terminal methionine is added to component (A) and/or prior to aprotein or peptide sequence component (B), (C), (D) and/or (E).

Preferably, component (A) and component (B) are linked by chemicalcoupling in any suitable manner known in the art, such as cross-linkingmethods. However, attention is drawn to the fact that many knownchemical cross-linking methods are non-specific, i.e., they do notdirect the point of coupling to any particular site on the carriermoiety or cargo moiety. Thus, the use of non-specific cross-linkingagents may attack functional sites or sterically block active sites,rendering the fused components of the inventive transporter cargoconjugate molecule biologically inactive. It is referred to theknowledge of the skilled artisan to block potentially reactice groups byusing appropriate protecting groups. Alternatively, the use of thepowerful and versatile oxime and hydrazone ligation techniques, whichare chemo-selective entities that can be applied for the cross-linkingof component (A) to component (B), may be employed. This linkingtechnology is described e.g. by Rose et al. (1994), JACS 116, 30. Ifpresent, further components (C), (D), (E) etc., as mentioned above, canbe chemically coupled in an analogous manner to one another or tocomponent (A) and/or (B).

Coupling specificity can be increased by direct chemical coupling to afunctional group found only once or a few times in component (A), whichfunctional group is to be cross-linked to the organic molecule ofcomponent (B). As an example, the cystein thiol group may be used, ifjust one cystein residue is present on component (A) of the inventivetransporter cargo conjugate molecule. Also, for example, if a conjugatemolecule component (A) contains no lysine residues, a cross-linkingreagent specific for primary amines will be selective for the aminoterminus of component (A). Alternatively, cross-linking may also becarried out via the side chain of a glutamic acid residue placed at theN-terminus of the peptide such that a amide bond can be generatedthrough its side-chain. Therefore, it may be advantageous to link aglutamic acid residue to the N-terminus of component (A) of theinventive transporter cargo conjugate molecule. However, if a cysteineresidue is to be introduced into component (A), introduction at or nearits N- or C-terminus is preferred. Conventional methods are availablefor such amino acid sequence alterations based on modifications ofcomponent (A) by either adding one or more additional amino acids, e.g.inter alia an cystein residue, to the translocation sequence or bysubstituting at least one residue of the translocation sequence(s) beingcomprised in component (A). In case a cystein side chain is used forcoupling purposes, component (A) of the inventive transporter cargoconjugate molecule has preferably one cystein residue. Any secondcystein residue should preferably be avoided and can, eventually, bereplaced when they occur in component (A) of the inventive transportercargo conjugate molecule. When a cysteine residue is replaced in theoriginal translocation sequence to be used as or as part of component(A), it is typically desirable to minimize resulting changes incomponent (A) peptide folding. Changes in component (A) folding areminimized when the replacement is chemically and sterically similar tocysteine. Therefore, serine is preferred as a replacement for cystein.

Coupling of the two constituents of the inventive transporter cargoconjugate molecule can be accomplished via a coupling or conjugatingagent including standard peptide synthesis coupling reagents such asHOBt, HBTU, DICI, TBTU. There are several intermolecular cross-linkingreagents which can be utilized, see for example, Means and Feeney,Chemical Modification of Proteins, Holden-Day, 1974, pp. 39-43. Amongthese reagents are, for example, N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) orN,N′-(1,3-phenylene)bismaleimide; N,N′-ethylene-bis-(iodoacetamide) orother such reagent having 6 to 11 carbon methylene bridges; and1,5-difluoro-2,4-dinitrobenzene. Other cross-linking reagents useful forthis purpose include: p,p′-difluoro-m,m′-dinitrodiphenylsulfone;dimethyl adipimidate; phenol-1,4-disulfonylchloride;hexamethylenediisocyanate or diisothiocyanate, orazophenyl-p-diisocyanate; glutaraldehyde and disdiazobenzidine.Cross-linking reagents may be homobifunctional, i.e., having twofunctional groups that undergo the same reaction. A preferredhomobifunctional cross-linking reagent is bismaleimidohexane (BMH). BMHcontains two maleimide functional groups, which react specifically withsulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). Thetwo maleimide groups are connected by a hydrocarbon chain. Therefore,BMH is useful for irreversible cross-linking of proteins (orpolypeptides) that contain cysteine residues. Cross-linking reagents mayalso be heterobifunctional. Heterobifunctional cross-linking agents havetwo different functional groups, for example an amine-reactive group anda thiol-reactive group, that will cross-link two proteins having freeamines and thiols, respectively. Examples of heterobifunctionalcross-linking agents areSuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and succinimide4-(p-maleimidophenyl)butyrate (SMPB), an extended chain analog of MBS.The succinimidyl group of these cross-linkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue. Because cross-linking reagents often havelow solubility in water, a hydrophilic moiety, such as a sulfonategroup, may be added to the cross-linking reagent to improve its watersolubility. Sulfo-MBS and sulfo-SMCC are examples of cross-linkingreagents modified for water solubility. Many cross-linking reagentsyield a conjugate that is essentially non-cleavable under cellularconditions. Therefore, some cross-linking reagents contain a covalentbond, such as a disulfide, that is cleavable under cellular conditions.For example, Traut's reagent, dithiobis (succinimidylpropionate) (DSP),and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) are well-knowncleavable cross-linkers. The use of a cleavable cross-linking reagentpermits the cargo moiety component (B), (C), (D) and/or (E) to separatefrom the novel transporter construct component (A) after delivery intothe target cell. For this purpose, direct disulfide linkage may also beuseful. Chemical cross-linking may also include the use of spacer arms.Spacer arms provide intramolecular flexibility or adjust intramoleculardistances between conjugated moieties and thereby may help preservebiological activity. A spacer arm may be in the form of a protein (orpolypeptide) moiety that includes spacer amino acids, e.g. proline.Alternatively, a spacer arm may be part of the cross-linking reagent,such as in “long-chain SPDP” (Pierce Chem. Co., Rockford, Ill., cat. No.21651 H). Numerous cross-linking reagents, including the ones discussedabove, are commercially available. Detailed instructions for their useare readily available from the commercial suppliers. A general referenceon protein cross-linking and conjugate preparation is: Wong, Chemistryof Protein Conjugation and Cross-Linking, CRC Press (1991).

According to another preferred embodiment, an inventive transportercargo conjugate molecule as defined above may comprise or consist of atleast one variant and/or fragment of the above defined inventivetransporter cargo conjugate molecules. Preferably, variants and/orfragments of the above defined inventive transporter cargo conjugatemolecules retain biological activity of the inventive transporter cargoconjugate molecules as disclosed above. Functionality of such fragmentsor variants may be tested by various tests, e.g. transfection efficacy,correct expression of proteins encoded by cargo nucleic acids, or bybiophysical methods, e.g. spectroscopy, computer modeling, structuralanalysis, etc. similar as described above for inventive noveltransporter constructs according to generic formula (I)

Even more preferably, the above defined inventive transporter cargoconjugate molecules comprise or consist of at least one variant (and/orfragment), particularly if the single components are protein or peptidesequences. In the context of the invention such variants (and/orfragments) of the above defined inventive transporter cargo conjugatemolecules may have a sequence identity to their native sequences, e.g. afragment or a variant of the above defined inventive transporter cargoconjugate molecule to its native transporter cargo conjugate moleculesequence, of at least 70%, 80% or 85%, preferably at least 90%, morepreferably at least 95% and most preferably at least 99% over the wholelength of the native inventive transporter cargo conjugate molecule.

A “fragment” of the above defined inventive transporter cargo conjugatemolecules, particularly if the single components are protein or peptidesequences, is preferably to be understood as a truncated sequencethereof, i.e. an amino acid sequence of the above defined inventivetransporter cargo conjugate molecule, which is N-terminally,C-terminally ally and/or intrasequentially truncated compared to theamino acid sequence of the native inventive transporter cargo conjugatemolecule as defined above.

A “variant” of the above defined inventive transporter cargo conjugatemolecule preferably comprises a sequence wherein the amino acid sequenceof the inventive transporter cargo conjugate molecule variant differsfrom the native sequence of the above defined inventive transportercargo conjugate molecule in one or more mutation(s), such as one or moresubstituted, (or, if necessary, inserted and/or deleted) amino acid(s).Preferably, variants of the above defined inventive transporter cargoconjugate molecules have the same biological function or specificactivity compared to the full-length native transporter cargo conjugatemolecules as defined above. Preferably, a variant may comprise about 1to 100, 1 to 50, 1 to 20, preferably 1 to 10 and more preferably 1 to 5,4, 3, 2 or 1 amino acid alterations within the above meaning. Suchalterations may comprise inter alia modifications of amino acids asdefined above, introduction of labels into amino acids as defined above,substituting an amino acid with any of the (modified or labelled) aminoacids mentioned herein, deletions or insertions of amino acids. Variantsas defined herein furthermore preferably comprise conservative aminoacid substitutions, preferably such as already defined above.

According to a third aspect, the present invention furthermore providesa pharmaceutical composition, the pharmaceutical composition preferablycomprising the inventive transporter cargo conjugate molecule as definedabove, and optionally a pharmaceutically acceptable carrier and/orvehicle, or any excipient, buffer, stabilizer or other materials wellknown to those skilled in the art.

As a first ingredient, the inventive pharmaceutical compositioncomprises the inventive transporter cargo conjugate molecule as definedabove, i.e. an inventive transporter cargo conjugate molecule,comprising as a component (A) the inventive novel transporter constructaccording to generic formula (I) or according to any of subformulas(Ia), (Ib), (Ic), (Id), (Ie), or (If) above, and as a component (B) aneffector molecule, selected from proteins or peptides, such astherapeutically active proteins and peptides, protein kinase inhibitors,particularly inhibitors of the protein kinase c-Jun amino terminalkinase, antigens, antibodies, apoptotic factors, proteases implicated inpathological states, preferably peptidic protease inhibitors,BH3-domains BH3-only proteins, or selected from nucleic acids, siRNAs,or from cytotoxic agents, small organic compounds, etc. Optionally, theinventive transporter cargo conjugate molecule as defined above mayfurthermore contain additional components (C), (D), and/or (E), etc.

As a second ingredient, the inventive pharmaceutical composition may ormay not comprise a pharmaceutically acceptable carrier and/or vehicle.In the context of the present invention, a pharmaceutically acceptablecarrier typically includes the liquid or non-liquid basis of theinventive inventive pharmaceutical composition. If the inventivepharmaceutical composition is provided in liquid form, the carrier willtypically be pyrogen-free water; isotonic saline or buffered (aqueous)solutions, e.g. phosphate, citrate etc. buffered solutions. Particularlyfor injection of the inventive inventive pharmaceutical composition,water or preferably a buffer, more preferably an aqueous buffer, may beused, containing a sodium salt, preferably at least 50 mM of a sodiumsalt, a calcium salt, preferably at least 0.01 mM of a calcium salt, andoptionally a potassium salt, preferably at least 3 mM of a potassiumsalt. According to a preferred embodiment, the sodium, calcium and,optionally, potassium salts may occur in the form of their halogenides,e.g. chlorides, iodides, or bromides, in the form of their hydroxides,carbonates, hydrogen carbonates, or sulfates, etc. Without being limitedthereto, examples of sodium salts include e.g. NaCl, NaI, NaBr, Na₂CO₃,NaHCO₃, Na₂SO₄, examples of the optional potassium salts include e.g.KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, and examples of calcium salts includee.g. CaCl₂, CaI₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂. Furthermore, organicanions of the aforementioned cations may be contained in the buffer.According to a more preferred embodiment, the buffer suitable forinjection purposes as defined above, may contain salts selected fromsodium chloride (NaCl), calcium chloride (CaCl₂) and optionallypotassium chloride (KCl), wherein further anions may be presentadditional to the chlorides. CaCl₂ can also be replaced by another saltlike KCl. Typically, the salts in the injection buffer are present in aconcentration of at least 50 mM sodium chloride (NaCl), at least 3 mMpotassium chloride (KCl) and at least 0.01 mM calcium chloride (CaCl₂).The injection buffer may be hypertonic, isotonic or hypotonic withreference to the specific reference medium, i.e. the buffer may have ahigher, identical or lower salt content with reference to the specificreference medium, wherein preferably such concentrations of the aforementioned salts may be used, which do not lead to damage of cells due toosmosis or other concentration effects. Reference media are e.g. liquidsoccurring in “in vivo” methods, such as blood, lymph, cytosolic liquids,or other body liquids, or e.g. liquids, which may be used as referencemedia in “in vitro” methods, such as common buffers or liquids. Suchcommon buffers or liquids are known to a skilled person. Ringer-Lactatesolution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well for the inventivepharmaceutical composition, which are suitable for administration to apatient to be treated. The term “compatible” as used here means thatthese constituents of the inventive pharmaceutical composition arecapable of being mixed with the inventive transporter cargo conjugatemolecule as defined above in such a manner that no interaction occurswhich would substantially reduce the pharmaceutical effectiveness of theinventive pharmaceutical composition under typical use conditions.Pharmaceutically acceptable carriers, fillers and diluents must, ofcourse, have sufficiently high purity and sufficiently low toxicity tomake them suitable for administration to a person to be treated. Someexamples of compounds which can be used as pharmaceutically acceptablecarriers, fillers or constituents thereof are sugars, such as, forexample, lactose, glucose and sucrose; starches, such as, for example,corn starch or potato starch; cellulose and its derivatives, such as,for example, sodium carboxymethylcellulose, ethylcellulose, celluloseacetate; powdered tragacanth; malt; gelatin; tallow; solid glidants,such as, for example, stearic acid, magnesium stearate; calcium sulfate;vegetable oils, such as, for example, groundnut oil, cottonseed oil,sesame oil, olive oil, corn oil and oil from theobroma; polyols, suchas, for example, polypropylene glycol, glycerol, sorbitol, mannitol andpolyethylene glycol; alginic acid.

The inventive pharmaceutical composition may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term parenteralas used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional, intracranial, transdermal, intradermal,intrapulmonal, intraperitoneal, intracardial, intraarterial, andsublingual injection or infusion techniques.

Preferably, the inventive pharmaceutical composition may be administeredby parenteral injection, more preferably by subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, intracranial, transdermal,intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, and sublingual injection or via infusion techniques.Sterile injectable forms of the inventive pharmaceutical compositionsmay be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1.3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents that arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation of the inventivepharmaceutical composition.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will preferably be in theform of a parenterally acceptable aqueous solution which is pyrogen-freeand has suitable pH, isotonicity and stability. Those of relevant skillin the art are well able to prepare suitable solutions using, forexample, isotonic vehicles such as Sodium Chloride Injection, Ringer'sInjection, Lactated Ringer's Injection. Preservatives, stabilizers,buffers, antioxidants and/or other additives may be included, asrequired. Whether it is a polypeptide, peptide, or nucleic acidmolecule, other pharmaceutically useful compound according to thepresent invention that is to be given to an individual, administrationis preferably in a “prophylactically effective amount” or a“therapeutically effective amount” (as the case may be), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated.

The inventive pharmaceutical composition as defined above may also beadministered orally in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient, i.e. the inventivetransporter cargo conjugate molecule as defined above, is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

The inventive pharmaceutical composition may also be administeredtopically, especially when the target of treatment includes areas ororgans readily accessible by topical application, e.g. includingdiseases of the skin or of any other accessible epithelial tissue.Suitable topical formulations are readily prepared for each of theseareas or organs. For topical applications, the inventive pharmaceuticalcomposition may be formulated in a suitable ointment, containing theinventive immunostimulatory composition, particularly its components asdefined above, suspended or dissolved in one or more carriers. Carriersfor topical administration include, but are not limited to, mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, theinventive pharmaceutical composition can be formulated in a suitablelotion or cream. In the context of the present invention, suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

In this context, prescription of treatment, e.g. decisions on dosageetc. when using the above pharmaceutical composition is typically withinthe responsibility of general practitioners and other medical doctors,and typically takes account of the disorder to be treated, the conditionof the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in REMINGTON'SPHARMACEUTICAL SCIENCES, 16th edition, Osol, A. (ed), 1980. Accordingly,the inventive pharmaceutical composition typically comprises a “safe andeffective amount” of the components of the inventive pharmaceuticalcomposition, particularly of the inventive transporter cargo conjugatemolecule as defined above. As used herein, a “safe and effective amount”means an amount of the inventive transporter cargo conjugate molecule asdefined above that is sufficient to significantly induce a positivemodification of a disease or disorder as defined herein. At the sametime, however, a “safe and effective amount” is small enough to avoidserious side-effects, that is to say to permit a sensible relationshipbetween advantage and risk. The determination of these limits typicallylies within the scope of sensible medical judgment. A “safe andeffective amount” of the components of the inventive pharmaceuticalcomposition, particularly of the inventive transporter cargo conjugatemolecule as defined above, will furthermore vary in connection with theparticular condition to be treated and also with the age and physicalcondition of the patient to be treated, the body weight, general health,sex, diet, time of administration, rate of excretion, drug combination,the activity of the specific components (A), (B), (C), (D) and/or (E) ofthe inventive transporter cargo conjugate molecule as defined above, theseverity of the condition, the duration of the treatment, the nature ofthe accompanying therapy, of the particular pharmaceutically acceptablecarrier used, and similar factors, within the knowledge and experienceof the accompanying doctor. The inventive pharmaceutical composition maybe used for human and also for veterinary medical purposes, preferablyfor human medical purposes, as a pharmaceutical composition in generalor as a vaccine.

According to a specific embodiment, the inventive pharmaceuticalcomposition may be provided as a vaccine, e.g. if component (B) of theinventive transporter cargo conjugate molecule is a therpeuticallyactive protein such as a(n) (protein or peptide) antigen or antigenicfragment or any molecule as described above, which is suitable to elicitan immune response. Such an inventive vaccine is typically composed likethe inventive pharmaceutical composition and preferably supports aninnate and/or an adaptive immune response of the immune system of apatient to be treated, depending on the nature of the components (B),(C), (D) and/or (E) of the inventive transporter cargo conjugatemolecule as defined above. As an example, if any of these componentsprovides or encodes a(n) (protein or peptide) antigen or antigenicfragment, the vaccine typically will lead to an adaptive immune responsein the patient to be treated. Similarly, any of the further components(B), (C), (D) and/or (E) of the inventive transporter cargo conjugatemolecule as defined above may lead to an innate and/or adaptive immuneresponse.

The inventive vaccine may also comprise a pharmaceutically acceptablecarrier, adjuvant, and/or vehicle as defined above for the inventivepharmaceutical composition. In the specific context of the inventivevaccine, the choice of a pharmaceutically acceptable carrier isdetermined in principle by the manner in which the inventive vaccine isadministered. The inventive vaccine can be administered, for example,systemically or locally. Routes for systemic administration in generalinclude, for example, transdermal, oral, parenteral routes, includingsubcutaneous, intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,vaccines may be administered by an intradermal, subcutaneous, orintramuscular route. Inventive vaccines are therefore preferablyformulated in liquid (or sometimes in solid) form. The suitable amountof the inventive vaccine to be administered can be determined by routineexperiments with animal models. Such models include, without implyingany limitation, rabbit, sheep, mouse, rat, dog and non-human primatemodels. Preferred unit dose forms for injection include sterilesolutions of water, physiological saline or mixtures thereof. The pH ofsuch solutions should be adjusted to about 7.4. Suitable carriers forinjection include hydrogels, devices for controlled or delayed release,polylactic acid and collagen matrices. Suitable pharmaceuticallyacceptable carriers for topical application include those which aresuitable for use in lotions, creams, gels and the like. If the inventivevaccine is to be administered orally, tablets, capsules and the like arethe preferred unit dose form. The pharmaceutically acceptable carriersfor the preparation of unit dose forms which can be used for oraladministration are well known in the prior art. The choice thereof willdepend on secondary considerations such as taste, costs and storability,which are not critical for the purposes of the present invention, andcan be made without difficulty by a person skilled in the art.

The inventive vaccine can additionally contain one or more auxiliarysubstances in order to further increase its immunogenicity. Asynergistic action of the inventive transporter cargo conjugate moleculeas defined above and of an auxiliary substance, which may be optionallycontained in the inventive vaccine as described above, is preferablyachieved thereby. Depending on the various types of auxiliarysubstances, various mechanisms can come into consideration in thisrespect. For example, compounds that permit the maturation of dendriticcells (DCs), for example lipopolysaccharides, TNF-alpha or CD40 ligand,form a first class of suitable auxiliary substances. In general, it ispossible to use as auxiliary substance any agent that influences theimmune system in the manner of a “danger signal” (LPS, GP96, etc.) orcytokines, such as GM-CFS, which allow an immune response produced bythe immune-stimulating adjuvant according to the invention to beenhanced and/or influenced in a targeted manner. Particularly preferredauxiliary substances are cytokines, such as monokines, lymphokines,interleukins or chemokines, that further promote the innate immuneresponse, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,IL-31, IL-32, IL-33, INF-alpha, IFN-beta, INF-gamma, GM-CSF, G-CSF,M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.

Further additives which may be included in the inventive vaccine areemulsifiers, such as, for example, Tween®; wetting agents, such as, forexample, sodium lauryl sulfate; colouring agents; taste-impartingagents, pharmaceutical carriers; tablet-forming agents; stabilizers;antioxidants; preservatives.

The inventive vaccine can also additionally contain any furthercompound, which is known to be immune-stimulating due to its bindingaffinity (as ligands) to human Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

Another class of compounds, which may be added to an inventive vaccinein this context, may be CpG nucleic acids, in particular CpG-RNA orCpG-DNA. A CpG-RNA or CpG-DNA can be a single-stranded CpG-DNA (ssCpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA(ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleicacid is preferably in the form of CpG-RNA, more preferably in the formof single-stranded CpG-RNA (ss CpG-RNA). The CpG nucleic acid preferablycontains at least one or more (mitogenic) cytosine/guanine dinucleotidesequence(s) (CpG motif(s)). According to a first preferred alternative,at least one CpG motif contained in these sequences, that is to say theC (cytosine) and the G (guanine) of the CpG motif, is unmethylated. Allfurther cytosines or guanines optionally contained in these sequencescan be either methylated or unmethylated. According to a furtherpreferred alternative, however, the C (cytosine) and the G (guanine) ofthe CpG motif can also be present in methylated form.

According to a fourth aspect of the present invention, the inventivetransporter cargo conjugate molecule as defined above may be used (forthe preparation of a pharmaceutical composition or a vaccine, preferablyboth as defined herein) for the prophylaxis, treatment and/oramelioration of any of the diseases and disorders as defined herein,preferably for the prophylaxis, treatment and/or amelioration of canceror tumor diseases, including diseases caused by defective apoptosis,inflammatory diseases, infectious diseases, viral (infectious) diseases,diseases strongly related to JNK signalling, autoimmune disorders ordiseases, cardiovascular diseases, neuronal or neurodegenerativediseases, diseases of the liver, diseases of the spine, diseases of theuterus, major depressive disorders, non-chronic or chronic inflammatorydigestive diseases and hearing loss or diseases of the inner ear. Theinventive transporter cargo conjugate molecule may also be used (for thepreparation of a pharmaceutical composition) for use in tissuetransplantation either by treating the organs/tissue/cells to betransplanted or by treating the recipient of the organ/tissue/cells.

Prophylaxis, treatment and/or amelioration of a disease as definedherein typically include administration of a pharmaceutical compositionas defined above. The term “prophylaxis” is typically directed to theprevention of a disease as defined herein in a patient, preferably priorto manifestation of the disease in the patient. The term “treatment”generally refers to any treatment of a disease as defined herein in apatient, wherein the disease may have already been diagnosed or shall beprevented, i.e. prior, parallel and subsequent to manifestation of thedisease in the patient. The term “treatment”, used for example in theterm “treating a condition”, furthermore preferably means at least theadministration of a therapeutically effective amount of a therapeuticcompound to elicit a therapeutic effect. It does not necessarily imply“curing”, but rather having preferably at least some minimalphysiological effect upon a condition upon administration to a livingbody having such a condition. For example, treatment could encompassadministering an agent and the presence of that agent resulting in achange in the physiology of a recipient animal. Finally, the term“amelioration” preferably includes any modification of a disease asdefined herein, preferably a positive modification f the disease asdefined herein. The specific modification may be dependent on thedisease to be treated.

According to one approach, an inventive pharmaceutical composition, avaccine or an inventive transporter cargo conjugate molecule as definedabove may be administered directly to a patient using the administrationroutes as described above for pharmaceutical compositions.Alternatively, a pharmaceutical composition, a vaccine or an inventivetransporter cargo conjugate molecule as defined above may beadministered to a patient using an ex vivo approach, e.g. by introducingthe pharmaceutical composition, the vaccine or the inventive transportercargo conjugate molecule as defined above into cells, preferablyautologous cells, i.e. cells derived from the patient to be treated, andtransplanting these cells into the site of the patient to be treated,optionally subsequent to storing and/or culturing these cells prior totreatment.

According to one preferred embodiment, the inventive transporter cargoconjugate molecule, the inventive pharmaceutical composition or theinventive vaccine as defined above, may be used for (the preparation ofa medicament for) the prophylaxis, treatment and/or amelioration of e.g.cancer or tumor diseases, including diseases caused by defectiveapoptosis, preferably selected from acusticus neurinoma, anal carcinoma,astrocytoma, basalioma, Behcet's syndrome, bladder cancer, blastomas,bone cancer, brain metastases, brain tumors, brain cancer(glioblastomas), breast cancer (mamma carcinoma), Burkitt's lymphoma,carcinoids, cervical cancer, colon carcinoma, colorectal cancer, corpuscarcinoma, craniopharyngeomas, CUP syndrome, endometrial carcinoma, gallbladder cancer, genital tumors, including cancers of the genitourinarytract, glioblastoma, gliomas, head/neck tumors, hepatomas, histocyticlymphoma, Hodgkin's syndromes or lymphomas and non-Hodgkin's lymphomas,hypophysis tumor, intestinal cancer, including tumors of the smallintestine, and gastrointestinal tumors, Kaposi's sarcoma, kidney cancer,kidney carcinomas, laryngeal cancer or larynx cancer, leukemia,including acute myeloid leukaemia (AML), erythroleukemia, acute lymphoidleukaemia (ALL), chronic myeloid leukaemia (CML), and chroniclymphocytic leukaemia (CLL), lid tumor, liver cancer, liver metastases,lung carcinomas (=lung cancer=bronchial carcinoma), small cell lungcarcinomas and non-small cell lung carcinomas, and lung adenocarcinoma,lymphomas, lymphatic cancer, malignant melanomas, mammary carcinomas(=breast cancer), medulloblastomas, melanomas, meningiomas, Mycosisfungoides, neoplastic diseases neurinoma, oesophageal cancer,oesophageal carcinoma (=oesophageal cancer), oligodendroglioma, ovariancancer (=ovarian carcinoma), ovarian carcinoma, pancreatic carcinoma(=pancreatic cancer), penile cancer, penis cancer, pharyngeal cancer,pituitary tumour, plasmocytoma, prostate cancer (=prostate tumors),rectal carcinoma, rectal tumors, renal cancer, renal carcinomas,retinoblastoma, sarcomas, Schneeberger's disease, skin cancer, e.g.melanoma or non-melanoma skin cancer, including basal cell and squamouscell carcinomas as well as psoriasis, pemphigus vulgaris, soft tissuetumours, spinalioma, stomach cancer, testicular cancer, throat cancer,thymoma, thyroid carcinoma, tongue cancer, urethral cancer, uterinecancer, vaginal cancer, various virus-induced tumors such as, forexample, papilloma virus-induced carcinomas (e.g. cervicalcarcinoma=cervical cancer), adenocarcinomas, herpes virus-induced tumors(e.g. Burkitt's lymphoma, EBV-induced B-cell lymphoma, cervixcarcinoma), heptatitis B-induced tumors (hepatocell carcinomas), HTLV-1-and HTLV-2-induced lymphomas, vulval cancer, wart conditions orinvolvement, etc. In the present context, the terms “therapy” and“therapeutic” preferably mean to have at least some minimalphysiological effect upon being administered to a living body. Forexample, a physiological effect upon administering a “therapeutic”anti-tumor compound may be the inhibition of tumor growth, or decreasein tumor size, or prevention reoccurrence of the tumor. Preferably, inthe treatment of cancer or neoplastic disease, a compound which inhibitsthe growth of a tumor or decreased the size of the tumor or prevents thereoccurrence of the tumor would be considered therapeutically effective.The term “anti-tumor drug” therefore preferably means any therapeuticagent having therapeutic effect against a tumor, neoplastic disease orcancer.

According to an alternative preferred embodiment, the inventivetransporter cargo conjugate molecule, the inventive pharmaceuticalcomposition or the inventive vaccine as defined above, may be used for(the preparation of a medicament for) the prophylaxis, treatment, and/oramelioration of inflammatory diseases, such as inflammatory diseases ofthe lung or lung diseases, including Acute Respiratory Distress Syndrome(ARDS), or pulmonary fibrosis, inflammations of the tissue, including,without being limited thereto, formation of fibrous tissue, includingcystic fibrosis, meningitis, and graft rejection or transplant rejectionreactions, chronic illness involving the respiratory system, includingAsthma, chronic obstructive pulmonary disease (COPD), pneumonia, andpulmonary fibrosis.

According to an alternative preferred embodiment, the inventivetransporter cargo conjugate molecule, the inventive pharmaceuticalcomposition or the inventive vaccine as defined above, may be used for(the preparation of a medicament for) the prophylaxis, treatment, and/oramelioration of e.g. infectious diseases, preferably viral, retroviral,bacterial or protozoological infectious diseases. Such infectiousdiseases are typically selected from AIDS, anthrax, Japaneseencephalitis, bacterial infectious diseases such as miscarriage(prostate inflammation), anthrax, appendicitis, borreliosis, botulism,Camphylobacter, Chlamydia trachomatis (inflammation of the urethra,conjunctivitis), cholera, diphtheria, donavanosis, epiglottitis, typhusfever, gas gangrene, gonorrhoea, rabbit fever, Heliobacter pylori,whooping cough, climatic bubo, osteomyelitis, Legionnaire's disease,chicken-pox, condyloma acuminata, cytomegalic virus (CMV), dengue fever,early summer meningoencephalitis (ESME), Ebola virus, colds, fifthdisease, foot-and-mouth disease, herpes simplex type I, herpes simplextype II, herpes zoster, HSV, infectious diseases caused by parasites,protozoa or fungi, such as amoebiasis, bilharziosis, Chagas disease,Echinococcus, fish tapeworm, fish poisoning (Ciguatera), fox tapeworm,athlete's foot, canine tapeworm, candidosis, yeast fungus spots,scabies, cutaneous Leishmaniosis, lambliasis (giardiasis), lice,malaria, microscopy, onchocercosis (river blindness), fungal diseases,bovine tapeworm, schistosomiasis, porcine tapeworm, toxoplasmosis,trichomoniasis, trypanosomiasis (sleeping sickness), visceralLeishmaniosis, nappy/diaper dermatitis or miniature tapeworm, infectiouserythema, influenza, Kaposi's sarcoma, Lassa fever, Leishmaniasis,leprosy, listeriosis, Lyme borreliosis, malaria, Marburg virusinfection, measles, meningitis, including bacterial meningitis,molluscum contagiosum, mononucleosis, mumps, Mycoplasma hominis,neonatal sepsis (Chorioamnionitis), noma, Norwalk virus infection,otitis media, paratyphus, Pfeiffer's glandular fever, plague, pneumonia,polio (poliomyelitis, childhood lameness), pseudo-croup, rabies,Reiter's syndrome, Rocky Mountainspotted fever, Salmonella paratyphus,Salmonella typhus, SARS, scarlet fever, shingles, hepatitis, smallpox,soft chancre, syphilis, tetanus, three-day fever, tripper, tsutsugamushidisease, tuberculosis, typhus, vaginitis (colpitis), viral diseasescaused by cytomegalovirus (CMV), orthopox variola virus, orthopoxalastrim virus, parapox ovis virus, molluscum contagiosum virus, herpessimplex virus 1, herpes simplex virus 2, herpes B virus, varicellazoster virus, pseudorabies virus, human cytomegaly virus, human herpesvirus 6, human herpes virus 7, Epstein-Barr virus, human herpes virus 8,hepatitis B virus, chikungunya virus, O'nyong'nyong virus, rubivirus,hepatitis C virus, GB virus C, West Nile virus, dengue virus, yellowfever virus, louping ill virus, St. Louis encephalitis virus, Japan Bencephalitis virus, Powassan virus, FSME virus, SARS, SARS-associatedcorona virus, human corona virus 229E, human corona virus Oc43,Torovirus, human T cell lymphotropic virus type I, human T celllymphotropic virus type II, HIV (AIDS), i.e. human immunodeficiencyvirus type 1 or human immunodeficiency virus type 2, influenza virus,Lassa virus, lymphocytic choriomeningitis virus, Tacaribe virus, Juninvirus, Machupo virus, Borna disease virus, Bunyamwera virus, Californiaencephalitis virus, Rift Valley fever virus, sand fly fever virus,Toscana virus, Crimean-Congo haemorrhagic fever virus, Hazara virus,Khasan virus, Hantaan virus, Seoul virus, Prospect Hill virus, Puumalavirus, Dobrava Belgrade virus, Tula virus, sin nombre virus, LakeVictoria Marburg virus, Zaire Ebola virus, Sudan Ebola virus, IvoryCoast Ebola virus, influenza virus A, influenza virus B, influenzaviruses C, parainfluenza virus, measles virus, mumps virus, respiratorysyncytial virus, human metapneumovirus, vesicular stomatitis Indianavirus, rabies virus, Mokola virus, Duvenhage virus, European batlyssavirus 1+2, Australian bat lyssavirus, adenoviruses A-F, humanpapilloma viruses, condyloma virus 6, condyloma virus 11, polyomaviruses, adeno-associated virus 2, rotaviruses, or orbiviruses,Varicella including Varizella zoster, and malaria virus, viralinfectious diseases such as AIDS, infectious diseases caused byCondyloma acuminata, hollow warts, Dengue fever, three-day fever, Ebolavirus, cold, early summer meningoencephalitis (FSME), flu, shingles,hepatitis, herpes simplex type I, herpes simplex type II, Herpes zoster,influenza, Japanese encephalitis, Lassa fever, Marburg virus, warts,West Nile fever, yellow fever, etc.

According to another preferred embodiment, the inventive transportercargo conjugate molecule, the inventive pharmaceutical composition orthe inventive vaccine, may be used for (the preparation of a medicamentfor) the prophylaxis, treatment, and/or amelioration of diseasesstrongly related to JNK signaling in a subject. Such diseases ordisorders strongly related to JNK signaling in a subject, without beinglimited thereto, are preferably selected from autoimmune disorders,cardiovascular diseases, cancer or tumor diseases as defined above,diabetes, including diabetes type 1 or type 2, inflammatory diseases asdefined above, hair loss, including Alopecia areata, diseases of thelung, neuronal or neurodegenerative diseases, diseases of the liver,diseases of the spine, diseases of the uterus, (viral) infectiousdiseases and depressive disorders. In the case of diseases or disordersstrongly related to JNK signaling the term “amelioration” may includethe suppression of expression of JNK when it is over-expressed, and/orthe suppression of phosphorylation of c-jun, ATF2 or NFAT4 in any of theabove diseases, for example, by using at least one JNK inhibitorsequence as defined herein coupled to the inventive novel transportermolecule within the above definitions, as a competitive inhibitor of thenatural c-jun, ATF2 and NFAT4 binding site in a cell. In this specificcontext, the term “modulate” also includes suppression of hetero- andhomomeric complexes of transcription factors made up of, without beinglimited thereto, c-jun, ATF2, or NFAT4 and their related partners, suchas for example the AP-1 complex that is made up of c-jun, AFT2 andc-fos. When a disease or disorder strongly related to JNK signaling asdefined above is associated with JNK overexpression, such suppressiveJNK inhibitor sequences can be introduced to a cell. In some instances,“modulate” in the context of diseases or disorders strongly related toJNK signaling may also include the increase of JNK expression, forexample by use of an IB peptide-specific antibody that blocks thebinding of an IB-peptide to JNK, thus preventing JNK inhibition by theIB-related peptide. Prevention and/or treatment of a subject with thepharmaceutical composition as disclosed above may be typicallyaccomplished by administering (in vivo) an (“therapeutically effective”)amount of said pharmaceutical composition to a subject, wherein thesubject may be e.g. any mammal, e.g. a human, a primate, mouse, rat,dog, cat, cow, horse or pig.

The term “therapeutically effective” means that the active component ofthe pharmaceutical composition is of sufficient quantity to amelioratethe disease or disorder strongly related to JNK signaling as definedabove. Further example may be found for the other diseases mentionedherein.

Accordingly, the inventive transporter cargo conjugate molecule, theinventive pharmaceutical composition or the inventive vaccine, may beused for (the preparation of a medicament for) the prophylaxis,treatment, and/or amelioration of autoimmune disorders or diseases.Autoimmune disorders or diseases can be broadly divided into systemicand organ-specific or localised autoimmune disorders, depending on theprincipal clinico-pathologic features of each disease. Autoimmunediseases may be divided into the categories of systemic syndromes,including systemic lupus erythematosus (SLE), Sjögren's syndrome,Scleroderma, Rheumatoid Arthritis and polymyositis or local syndromeswhich may be endocrinologic (type I diabetes (Diabetes mellitus Type 1),Hashimoto's thyroiditis, Addison's disease etc.), dermatologic(pemphigus vulgaris), haematologic (autoimmune haemolytic anaemia),neural (multiple sclerosis) or can involve virtually any circumscribedmass of body tissue. The autoimmune diseases to be treated may beselected from the group consisting of type I autoimmune diseases or typeII autoimmune diseases or type III autoimmune diseases or type IVautoimmune diseases, such as, for example, multiple sclerosis (MS),rheumatoid arthritis, diabetes, type I diabetes (Diabetes mellitus Type1), chronic polyarthritis, Basedow's disease, autoimmune forms ofchronic hepatitis, colitis ulcerosa, type I allergy diseases, type IIallergy diseases, type III allergy diseases, type IV allergy diseases,fibromyalgia, hair loss, Bechterew's disease, Crohn's disease,Myasthenia gravis, neurodermitis, Polymyalgia rheumatica, progressivesystemic sclerosis (PSS), Reiter's syndrome, rheumatic arthritis,psoriasis, vasculitis, etc, or type II diabetes. While the exact mode asto why the immune system induces an immune reaction against autoantigenshas not been elucidated so far, there are several findings with regardto the etiology. Accordingly, the autoreaction may be due to a T-Cellbypass. A normal immune system requires the activation of B-cells byT-cells before the former can produce antibodies in large quantities.This requirement of a T-cell can be by-passed in rare instances, such asinfection by organisms producing super-antigens, which are capable ofinitiating polyclonal activation of B-cells, or even of T-cells, bydirectly binding to the B-subunit of T-cell receptors in a non-specificfashion. Another explanation deduces autoimmune diseases from aMolecular Mimicry. An exogenous antigen may share structuralsimilarities with certain host antigens; thus, any antibody producedagainst this antigen (which mimics the self-antigens) can also, intheory, bind to the host antigens and amplify the immune response. Themost striking form of molecular mimicry is observed in Group Abeta-haemolytic streptococci, which shares antigens with humanmyocardium, and is responsible for the cardiac manifestations ofrheumatic fever.

The inventive transporter cargo conjugate molecule, the inventivepharmaceutical composition or the inventive vaccine, may also be usedfor (the preparation of a medicament for) the prophylaxis, treatment,and/or amelioration of cardiovascular diseases, preferably selected fromheart diseases and coronary heart diseases, arteriosclerosis, apoplexy,dilatation of the abdominal aorta, such as infrarenal aneurismhypertension, and myocardial infarction.

Additionally, the inventive transporter cargo conjugate molecule, theinventive pharmaceutical composition or the inventive vaccine, may beused for (the preparation of a medicament for) the prophylaxis,treatment, and/or amelioration of neuronal or neurodegenerative diseasesselected from, without being limited thereto, Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis (ALS), dystonia,epilepsy, optic nerve disease, including glaucoma, eye infection,multiple sclerosis, meningitis, neuronal diseases caused by or disordersor diseases or disorders of the nervous system, including the “cutting”or disruption of axons, such as axotomy, pain, particularly neuropathicpain, stroke, including ischemic stroke, and viral encephalopathy.

The inventive transporter cargo conjugate molecule, the inventivepharmaceutical composition or the inventive vaccine, may also be usedfor (the preparation of a medicament for) the prophylaxis, treatment,and/or amelioration of diseases of the liver selected from, withoutbeing limited thereto, Hepatitis, and hepatotoxicity.

Additionally, the inventive transporter cargo conjugate molecule, theinventive pharmaceutical composition or the inventive vaccine, may beused for (the preparation of a medicament for) the prophylaxis,treatment, and/or amelioration of diseases of the spine, selected from,without being limited thereto, disc herniation.

According to one preferred embodiment, the inventive transporter cargoconjugate molecule, the inventive pharmaceutical composition or theinventive vaccine, may be used for (the preparation of a medicament for)the prophylaxis, treatment, and/or amelioration of diseases of theuterus selected from, without being limited thereto, endometriosis.

According to another preferred embodiment, the inventive transportercargo conjugate molecule, the inventive pharmaceutical composition orthe inventive vaccine, may be used for (the preparation of a medicamentfor) the prophylaxis, treatment, and/or amelioration of depressivedisorders selected from, without being limited thereto, major depressivedisorders, also known as major depression, unipolar depression, clinicaldepression, or simply depression, bipolar disorders, mania and maniacdepression.

According to a further preferred embodiment the inventive transportercargo conjugate molecule, the inventive pharmaceutical composition orthe inventive vaccine, may be used for (the preparation of a medicamentfor) the prophylaxis, treatment, and/or amelioration of non-chronic orchronic inflammatory digestive diseases in a subject. The term“non-chronic or chronic inflammatory digestive disease” as used hereintypically denotes non-chronic or chronic inflammatory diseases thatpertain to the gastrointestinal tract. This includes diseases of theesophagus, stomach, first, second, third and fourth part of theduodenum, jejunum, ileum, the ileo-cecal complex, large intestine,(ascending, transverse and descending colon) sigmoid colon and rectum.Preferably included in this respect are chronic inflammatory digestivediseases, which are characterized by an inflammation of the colon, suchas colitis, including e.g. Colitis ulcerosa (ulcerative colitis), MorbusCrohn (Crohn's disease), diversion colitis, ischemic colitis, infectiouscolitis, fulminant colitis, chemical colitis, microscopic colitis,lymphocytic colitis, collageneous colitis, indeterminate colitis andatypical colitis, etc.

In the context of the above, the invention relates also to the use ofthe inventive transporter cargo conjugate molecule the inventivepharmaceutical composition or the inventive vaccine, for theprophylaxis, treatment, and/or amelioration of diseases or disorders asmentioned herein. It also includes in particular the use of theinventive transporter cargo conjugate molecule, the inventivepharmaceutical composition or the inventive vaccine, for inoculation orthe use of these components as an inoculant. According to oneparticularly preferred embodiment of the present invention, such amethod for prophylaxis, treatment, and/or amelioration of theabove-mentioned diseases or disorders, or an inoculation method forpreventing the above-mentioned diseases, typically comprisesadministering the described inventive transporter cargo conjugatemolecule, pharmaceutical composition or vaccine to a patient in needthereof (e.g. suffering from any of the above diseases or showingsymptoms thereof), in particular to a human being, preferably in a “safeand effective amount” and in one of the above formulations as describedabove. The administration mode also may be as described above forinventive pharmaceutical compositions or vaccines.

According to a fifth aspect of the present invention, the inventivepharmaceutical composition, the inventive vaccine, the inventivetransporter cargo conjugate molecule as defined above, the inventivenovel transporter construct according to generic formula (I) oraccording to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If)above, or variants or fragments thereof within the above definitions,may be utilized as a medicament. Such a medicament may be apharmaceutical composition or a vaccine as shown above. It may beutilized in medical applications in general, preferably for any of theprophylaxis, treatment, and/or amelioration of diseases or disorders asmentioned herein.

According to a sixth aspect of the present invention, the inventivetransporter cargo conjugate molecule and preferably the inventive noveltransporter construct according to generic formula (I) or according toany of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If) above may beutilized for the transport of any cargo molecule (preferably as definedherein) into cells or tissue of a patient to be treated. In thiscontext, the cargo molecule is preferably suitable for a therapy asmentioned herein, particularly for the prophylaxis, treatment, and/oramelioration of diseases or disorders as mentioned herein and may beselected from any cargo molecule suitable therefore, more preferablyfrom any cargo molecule as described above for any of components (B),(C), (D) and/or (E) of the inventive transporter cargo conjugatemolecule. Thereby, the inventive novel transporter construct accordingto generic formula (I) or according to any of subformulas (Ia), (Ib),(Ic), (Id), (Ie), or (If) above may be coupled to such a cargo moleculeusing any of the coupling methods described for components (A), (B),(C), (D) and/or (E) above.

According to a particularly preferred embodiment of this aspect, theinventive transporter cargo conjugate molecule and more preferably theinventive novel transporter construct according to generic formula (I)or according to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If)above, may be utilized for the transport of any cargo molecule asmentioned herein, e.g. as described above for any of components (B),(C), (D) and/or (E), into cells, preferably into blood cells, morepreferably into white blood cells or preferably into neuronal cells. Thetransport may be effected in vitro, in vivo and/or ex vivo. Preferably,the transport is effected into the respective cells of a patient to betreated. Such a direction of the inventive transporter cargo conjugatemolecule and more preferably of the inventive novel transporterconstruct according to generic formula (I) or according to any ofsubformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If) conjugated to a cargoas mentioned above is particularly suitable in the treatment ofinflammatory diseases as mentioned above. For this purpose, theinventive transporter cargo conjugate molecule and more preferably theinventive novel transporter construct according to generic formula (I)or according to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If)above, may be administered as described above for an inventivepharmaceutical composition or an inventive vaccine. Furthermore, theinventive transporter cargo conjugate molecule and more preferably theinventive novel transporter construct according to generic formula (I)or according to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If)above, may be formulated as an inventive pharmaceutical composition oran inventive vaccine as described above for the purpose ofadministration. Administration routes are as defined above for aninventive pharmaceutical composition or an inventive vaccine.Alternatively, the inventive transporter cargo conjugate molecule andmore preferably the inventive novel transporter construct according togeneric formula (I) or according to any of subformulas (Ia), (Ib), (Ic),(Id), (Ie), or (If) above, may be administered directly without anyfurther formulation, i.e. “naked”. Likewise, administration routes arepreferably as described above for such a formulation.

According to a seventh aspect of the present invention, the inventivenovel transporter construct according to generic formula (I) oraccording to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If)above, the inventive transporter cargo conjugate molecule as definedabove or variants or fragments thereof within the above definitions, theinventive pharmaceutical composition or the inventive vaccine may beutilized in diagnosis as a diagnostic tool, e.g. in (in vivo or invitro) assays, e.g. in immunoassays, to detect, prognose, diagnose, ormonitor various conditions and disease states of disorders or diseasesmentioned.

As an example, immunoassay may be performed by a method comprisingcontacting a sample derived from a patient with an inventive transportercargo conjugate molecule as defined above, wherein component (B) and/orany of components (C), (D) and/or (E) of the inventive transporter cargoconjugate molecule may be directed against a component or compound, e.g.a a (cell) specific component or compound, contained in the sample. Sucha component (B) or any of components (C), (D) and/or (E) of theinventive transporter cargo conjugate molecule may be e.g. an antibodydirected to a (cell) specific component or compound of the sample,wherein such (cell) specific component or compound of the sample may bee.g. a compound or component as described above for any of components(B), (C), (D) and/or (E) as defined above. Contacting of the sample istypically carried out under conditions that immunospecific-binding mayoccur, and subsequently detecting or measuring the amount of anyimmunospecific-binding by the antibody. In a specific embodiment, anantibody specific for a (cell) specific component or compound of thesample, e.g. component (B), (C), (D) and/or (E) as defined above, may beused to analyze a tissue or serum sample from a patient for the presenceof such a component (B), (C), (D) and/or (E) as defined above or adisease associated therewith. Such diseases may include diseases ordisorders as described herein. The immunoassays that may be utilizedinclude, but are not limited to, competitive and non-competitive assaysystems using techniques such as Western Blots, radioimmunoassays (RIA),enzyme linked immunosorbent assay (ELISA), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,fluorescent immunoassays, complement-fixation assays, immunoradiometricassays, and protein-A immunoassays, etc.

Alternatively, (in vitro) assays may be performed by delivering theinventive pharmaceutical composition, a vaccine or the inventivetransporter cargo conjugate molecule as defined above or variants orfragments thereof within the above definitions to target cells typicallyselected from e.g. cultured animal cells, human cells ormicro-organisms, and to monitor the cell response by biophysical methodstypically known to a skilled person. The target cells typically usedtherein may be cultured cells (in vitro) or in vivo cells, i.e. cellscomposing the organs or tissues of living animals or humans, ormicroorganisms found in living animals or humans. Particularlypreferable in this context are so called markes or labels, which may becontained as a component (B) or any of components (C), (D) and/or (E) ofthe inventive transporter cargo conjugate molecule, wherein such labelsmay be as defined in general above for the inventive transporter cargoconjugate molecule, e.g.

According to a final aspect of the present invention, the presentinvention also provides kits, particularly kits of parts, comprising ascomponents alone or in combination, the inventive novel transporterconstruct according to generic formula (I) or according to any ofsubformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If) above, the inventivetransporter cargo conjugate molecule, the inventive pharmaceuticalcomposition and/or the inventive vaccine, and optionally technicalinstructions with information on the administration and dosage of thesecomponents. Such kits, preferably kits of parts, may be applied, e.g.,for or in any of the above mentioned applications or uses. The presentinvention additionally particularly provides the use of kits fordiagnostic or therapeutic purposes, particular for the treatment,prevention or monitoring of diseases or disorders as disclosed.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entirety.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features and advantages of the invention will beapparent from the following detailed description and claims.

EXAMPLES

The following examples are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

1. Preparation of (Peptidic) Transporter Constructs

The (peptidic) transporter constructs used in these examples wereprepared using solid phase synthesis as described above. The constructsused for identification of the minimal D-/L-pattern of transporterconstructs, which is sensitive to proteases were, inter alia, asfollows:

SEQ D-/L- Sequence ID No. Pattern (N-term to C-term) 123 all LRRRRRRRRR (reference sequence) 257 all D rrrrrrrrr 258 D/L rRrRrRrRr 259DD/LL rrRRrrRRr 260 DDD/LLL rrrRRRrrr (r₆R₃) 261 DDDD/LLLL rrrRRRRrr

The constructs used for uptake (internalization) experiments intovarious cells and cell lines were as follows:

Name Sequence (N-term to C-term) FITC-βA-L-TAT FITC-βA-RKKRQRRRFITC-βA-D-TAT FITC-βA-rrrqrrkkr FITC-βA-r₃-L-TAT FITC-βA-rKKRrQRRrFITC-βA-r₃-L-TATi FITC-βA-rRRQrRKKr FITC-βA-DAK (ctl) FITC-βA-DAK(no transporter sequence)

Subsequent to synthesis the constructs were purified, stored as 10 mMsolution in sterile water, and used as purified without any furthertreatment.

2. Identification of the Minimal D-/L-Pattern Conferring Sensibility toProteases (Proteinase K)

In order to identify the minimal D-/L-pattern of transporter constructs,which is sensitive to proteases but confers a sufficient long half lifein vivo, a quantitative degradation assay was carried out withProteinase K involving several transporter constructs with poly-Argsequences, each showing a different pattern of D- and L-amino acids. Thetransporter constructs used in this assay were prepared as describedunder example 1 above and termed 1 to 6.

SEQ  Sequence Protease ID No. Pattern (N-term to C-term) sensitivity 123all L RRRRRRRRR (reference + sequence) 257 all D rrrrrrrrr - 258 D/LrRrRrRrRr - 259 DD/LL rrRRrrRRr - 260 DDD/LLL rrrRRRrrr + (r₆R₃) 261DDDD/LLLL rrrRRRRrr +

The different pattern of the sequences is described using capitals andminor letters. The capitals in these sequences 1 to 6 (“R”-amino acids)refer to L-enatiomeric arginine (L-Arg) and the minor letters in thesesequences (“r”-amino acids) refer to D-enatiomeric arginine (L-Arg). Thesensitivity to Proteinase K was measured at time intervals t=0, 10 and40 minutes. The results of the quantitative degradation assay withProteinase K were then determined on the basis of samples taken at thesespecific time intervals. These samples were analyzed utilizing a massspectrometry analysis using starting material ion intensities asreference values. As a conclusion, when a stretch of 3 or more L-Arg ispresent in the sequence the peptide shows sensitivity to Proteinase Kand is degraded. Peptides with 2 or less L-Arg next to each other arenot degraded. These results are comparable to a sequence consistingentirely of D-enantiomeric amino acids, e.g. D-TAT (rrrqrrkkr; SEQ IDNO: 251) or D-JNKi, respectively (see also FIG. 1).

A further comparison was carried out using four transporter constructsof D-/L-TAT derivatives termed L-TAT (SEQ ID NO: 18), r₃-TAT (alsotermed r₃-L-Tat; SEQ ID NO: 20) r₃-TATi (also termed r₃-L-TATi; SEQ IDNO: 21), and D-TAT (SEQ ID NO: 251), each having a length of 9 aminoacids but a different D-/L-pattern. The different pattern of thesequences is described using capitals and minor letters. The capitals inthe sequences (“R”-amino acids) refer to L-enatiomeric arginine (L-Arg)and the minor letters in these sequences (“r”-amino acids) refer toD-enatiomeric arginine (L-Arg). The table shown in FIG. 2 illustratesthe amino acid sequences of these TAT derived transporter constructswith respect to their molecular weight (Mw) and pI-values (see FIG. 2).

The digestion was carried out in 10% and 50% human serum at 37° C. untilcomplete degradation of these TAT derived transporters construct. As canbe seen in FIG. 3, showing the results of the digestion of TAT derivedtransporter constructs, D-TAT (SEQ ID NO: 251) transporter constructsare protease resistant, whereas the L-TAT transporter construct (SEQ IDNO: 18) are degraded too early in vivo in order to effect an efficienttransport into cells. Only transporter constructs r3-TAT (also termedr3-L-Tat, SEQ ID NO: 20), r3-TATi (also termed r3-L-TATi, SEQ ID NO: 21)show a degradation within a suitable time limit, allowing to limit thein vivo stability for therapeutic applications.

A mass spectrometry analysis of the degradation of the transporterconstruct r3-L-TAT in human serum was carried out. As a result, r3-L-TAT(SEQ ID NO: 20) is C-terminally degraded up to single amino acid inhuman serum.

Furthermore, a digestion of TAT derived transporter constructs in 10%and 50% human serum at 37° C. was carried out until complete degradationof these TAT derived transporters under involvement of beta-Alanine(b-Ala) in protecting the peptides. Therefore, beta-Ala was added to theN-terminus of TAT derived transporter constructs L-TAT as L-TATi asalready described in FIG. 3. The results are shown in FIG. 4. As can beseen in FIG. 4 N-terminal protection of the transporter peptides withbeta-Alanine does not lead to a significant effect, i.e. an improvedstability.

Summarizing the above results with respect to their in vivo stabilityand functional half life the stability of the 4 TAT derived peptidestested in 10 and 50% HS is as follows: D-TAT (SEQ ID NO: 251)≤r3-L-TATi(SEQ ID NO: 21)>r3-L-TAT (SEQ ID NO: 20)>L-TAT (SEQ ID NO: 18). The lifetime of both r3-L-TAT (SEQ ID NO: 20) and r3-L-TATi (SEQ ID NO: 21) istherefore increased compared to L-TAT (SEQ ID NO: 18) and decreasedcompared to D-TAT (SEQ ID NO: 251). As serum constitutes ˜50% of bloodvolume, values obtained from samples containing 50% HS are the mostrelevant. UPLC-MS results furthermore suggest that the degradationinvolves exoproteases that cleave at the C-terminus of TAT.Carboxypeptidase N is assumed to be involved in the degradation as it isconstitutively active and present at high concentration (30□g/mL) in theblood. Moreover, CPN cleaves specifically C-terminal Lys or Arg frompeptides and proteins.

3. Uptake (Internalization) of Peptides into Cells and Measurement ofPeptide Internalization into Cells

In this experiment, the internalization (uptake) capacity ofFITC-labeled TAT derived transporter constructs in vitro was evaluatedwith a fluorescence plate reader in cell lines HL-60 (Leukemia).

3.1. Test Samples Used in the Experiments

The constructs used in this experiment were four different TAT derivedtransporter constructs (termed L-TAT (SEQ ID NO: 18), r3-TAT (alsotermed r3-L-Tat; SEQ ID NO: 20), r3-TATi (also termed r3-L-TATi; SEQ IDNO: 21), and D-TAT (SEQ ID NO: 251), each prepared as described above.These constructs have a length of 9 amino acids but a differentD-/L-pattern. Furthermore, the construct DAK was used as a control,which comprised no transporter sequence. The constructs wereN-terminally protected with beta-Alanine (βA) and labeled with FITC.

FITC-βA-L-TAT FITC-βA-RKKRQRRR FITC-βA-D-TAT FITC-βA-rrrqrrkkrFITC-βA-r₃-L-TAT FITC-βA-rKKRrQRRr FITC-βA-r₃-L-TATi FITC-βA-rRRQrRKKrFITC-βA-DAK (ctl) FITC-βA-DAK (no transporter sequence)

The constructs were prepared as described above, purified, stored as 10mM solution in sterile water, and used as purified without any furthertreatment.

3.2. Further Transporter Contructs TAT(s2-1)-TAT(s2-96)

Further transporter constructs TAT(s2-1)-TAT(s2-96) were prepared asdescribed above in general for inventive transporter constructs.Following sequences and protecting groups were used therefore duringsynthesis (bound to resin):

TATs2-1: D-Arg(Pmc)-Ala-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-2:D-Arg(Pmc)-Lys(Boc)-Ala-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-3:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Ala-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-4:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Ala-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-5:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Ala-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-6:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Ala-D-Arg(Pmc)-RESIN TATs2-7:D-Arg(Pmc)-Asp(OBut)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-8:D-Arg(Pmc)-Lys(Boc)-Asp(OBut)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-9:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Asp(OBut)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-10:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Asp(OBut)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-11:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Asp(OBut)-Arg(Pmc)-D-Arg(Pmc)-TATs2-RESIN TATs2-12:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Asp(OBut)-D-Arg(Pmc)-TATs2-RESIN TATs2-13:D-Arg(Pmc)-Glu(OBut)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-14:D-Arg(Pmc)-Lys(Boc)-Glu(OBut)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-15:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Glu(OBut)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-16:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Glu(OBut)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-17:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Glu(OBut)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-18:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Glu(OBut)-D-Arg(Pmc)-RESIN TATs2-19:D-Arg(Pmc)-Phe-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-20:D-Arg(Pmc)-Lys(Boc)-Phe-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-21:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Phe-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-22:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Phe-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-23:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Phe-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-24:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Phe-D-Arg(Pmc)-RESIN TATs2-25:D-Arg(Pmc)-Arg(Pmc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-26:D-Arg(Pmc)-Lys(Boc)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-27:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Lys(Boc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-28:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Arg(Pmc)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-29:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-30:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Lys(Boc)-D-Arg(Pmc)-RESIN TATs2-31:D-Arg(Pmc)-His(Trt)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-32:D-Arg(Pmc)-Lys(Boc)-His(Trt)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-33:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-His(Trt)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-34:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)His(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-35:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-His(Trt)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-36:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-His(Trt)-D-Arg(Pmc)-RESIN TATs2-37:D-Arg(Pmc)-Ile-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-38:D-Arg(Pmc)-Lys(Boc)-Ile-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-39:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Ile-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-40:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Ile-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-41:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Ile-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-42:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Ile-D-Arg(Pmc)-RESIN TATs2-43:D-Arg(Pmc)-Leu-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-44:D-Arg(Pmc)-Lys(Boc)-Leu-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-45:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Leu-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-46:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Leu-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-47:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Leu-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-48:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Leu-D-Arg(Pmc)-RESIN TATs2-49:D-Arg(Pmc)-Met-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-50:D-Arg(Pmc)-Lys(Boc)-Met-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-51:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Met-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-52:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Met-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-53:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Met-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-54:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Met-D-Arg(Pmc)-RESIN TATs2-55:D-Arg(Pmc)-Asn(Trt)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-56:D-Arg(Pmc)-Lys(Boc)-Asn(Trt)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-57:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Asn(Trt)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-58:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Asn(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-59:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Asn(Trt)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-60:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Asn(Trt)-D-Arg,(Pmc)-RESIN TATs2-61:D-Arg(Pmc)-Gln(Trt)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-62:D-Arg(Pmc)-Lys(Boc)-Gln(Trt)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-63:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Gln(Trt)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-64:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)Lys(Boc)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-65:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Gln(Trt)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-66:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Gln(Trt)-D-Arg(Pmc)-RESIN TATs2-67:D-Arg(Pmc)-Ser(But)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-68:D-Arg(Pmc)-Lys(Boc)-Ser(But)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-69:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Ser(But)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-70:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)Ser(But)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-71:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Ser(But)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-72:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Ser(But)-D-Arg(Pmc)-RESIN TATs2-73:D-Arg(Pmc)-Thr(But)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-74:D-Arg(Pmc)-Lys(Boc)-Thr(But)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-75:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Thr(But)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-76:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)Thr(But)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-77:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Thr(But)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-78:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Thr(But)-D-Arg(Pmc)-RESIN TATs2-79:D-Arg(Pmc)-Val-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-80:D-Arg(Pmc)-Lys(Boc)-Val-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-81:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Val-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-82:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Val-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-83:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Val-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-84:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Val-D-Arg(Pmc)-RESIN TATs2-85:D-Arg(Pmc)-Trp(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-86:D-Arg(Pmc)-Lys(Boc)-Trp(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-87:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Trp(Boc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-88:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)Trp(Boc)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-89:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Trp(Boc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-90:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Trp(Boc)-D-Arg(Pmc)-RESIN TATs2-91:D-Arg(Pmc)-Tyr(But)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-92:D-Arg(Pmc)-Lys(Boc)-Tyr(But)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-93:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Tyr(But)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-94:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)Tyr(But)-Arg(Pmc)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-95:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Tyr(But)-Arg(Pmc)-D-Arg(Pmc)-RESIN TATs2-96:D-Arg(Pmc)-Lys(Boc)-Lys(Boc)-Arg(Pmc)-D-Arg(Pmc)-Gln(Trt)-Arg(Pmc)-Tyr(But)-D-Arg(Pmc)-RESIN

3.3 Materials and Methods for Uptake Experiments

a) Cell Line:

The cell line used for this experiment was HL-60 (Ref CCL-240, ATCC, Lot116523)

b) Culture Medium and Plates

RPMI (Ref 21875-091, Invitrogen, Lot 8296) or DMEM (Ref 41965,Invitrogen, Lot 13481) complemented on May 5, 2008 with:

-   -   10% FBS (Ref A64906-0098, PAA, Lot A15-151): decomplemented at        56° C., 30 min, on Apr. 4, 2008.    -   1 mM Sodium Pyruvate (Ref 58636, Sigma, Lot 56K2386)    -   Penicillin (100 unit/ml)/Streptomycin (100 μg/ml) (Ref P4333,        Sigma, Lot 106K2321)

PBS 10× (Ref 70011, Invitrogen, Lot 8277): diluted to 1× with sterileH₂O

Trypsine-0.05% EDTA (Ref L-11660, PAA, Lot L66007-1194)

6 well culture plates (Ref 140675, Nunc, Lot 102613)

24 well culture plates (Ref 142475, Nunc, Lot 095849)

96 well culture plates (Ref 167008, Nunc, Lot 083310)

96 well plates for protein dosing (Ref 82.1581, Sarstedt)

96 well plates for fluorescence measurement (Ref 6005279, Perkin Elmer)

c) Solutions

Poly-D-lysine coating solution (Sigma P9011 Lot 095K5104): 25 μg/mlfinal diluted in PBS 1×

Acidic wash buffer: 0.2M Glycin, 0.15M NaCl, pH 3.0

Ripa lysis buffer: 10 mM NaH₂PO₄ pH 7.2, 150 mM NaCl, 1% Triton X-100, 1mM EDTA pH 8.0, 200 μM Na₃VO₂, 0.1% SDS, 1× protease inhibitor cocktail(Ref 11873580001, Roche, Lot 13732700)

d) Microscopy and Fluorescence Plate Reader

Cells were observed and counted using an inverted microscope (Axiovert40 CFL; Zeiss; 20×).

The fluorescence was read with the Fusion Alpha Plate reader (PerkinElmer).

e) Method

FITC marked peptide internalization was studied on suspension cells.Cells were plated into poly-DL-lysine coated dishes at a concentrationof 1×10⁶ cells/ml. Plates were then incubated for 24 h at 37° C., 5% CO,and 100% relative humidity prior to the addition of a knownconcentration of peptide. After peptide addition, the cells wereincubated 30 min, 1, 6 or 24 h at 37° C., 5% CO, and 100% relativehumidity. Cells were then washed twice with an acidic buffer (Glycin 0.2M, NaCl 0.15 M, pH 3.0) in order to remove the cell-surface adsorbedpeptide (see Kameyama et al., (2007), Biopolymers, 88, 98-107). Theacidic buffer was used as peptides rich in basic amino acids adsorbstrongly on the cell surfaces, which often results in overstimation ofinternalized peptide. The cell wash using an acidic buffer was thusemployed to remove the cell-surface adsorbed peptides. The acid wash wascarried out in determining cellular uptake of Fab/cell-permeatingpeptide conjugates, followed by two PBS washes. Cells were broken by theaddition of the RIPA lysis buffer. The relative amount of internalizedpeptide was then determined by fluorescence after backgroundsubstraction and protein content normalization.

The steps are thus: 1. Cell culture

-   -   2. Acidic wash and cellular extracts    -   3. Analysis of peptide internalization with a fluorescence plate        reader

f) Cell Culture and Peptide Treatment

-   -   (1) The 6 well culture plates are coated with 3 ml of Poly-D-Lys        (Sigma P9011; 25 μg/ml in PBS), the 24 well plates with 600 μl        and the 96 well plates with 125 μl and incubated for 4 h at 37°        C., CO, 5% and 100% relative humidity.    -   (2) After 4 hours the dishes were washed twice with 3.5 ml PBS,        700 μl or 150 μl PBS for the 6, 24 or 96 well plates,        respectively.    -   (3) The cells were plated into the dishes in 2.4 ml medium        (RPMI) at plating densities of 1,000,000 cells/ml for suspension        cells. After inoculation, the plates were incubated at 37° C.,        5% CO₂ and 100% relative humidity for 24 hours prior to the        addition of the peptide. Adherent cells should be at a density        of 90-95% the day of treatment and were plated in DMEM:

Surface of Nb Nb culture adherent suspension well (cm²) Medium cellscells 96 well 0.3 100-200 μl    8,000-30,000   100,000 24 well 2500-1000 μl   100,000-   500,000-   200,000 1,000,000 35 mm (P35)/ 102.4 ml   250,000- 2,400,000  6 well 2,100,000 60 mm (P60) 20 3.5 ml  15 * 10⁵ 1,000,000/ml 10 cm (P100) 60  10 ml 15-60 * 10⁵

-   -   (4) The cells were treated with the desired concentration of        FITC labeled peptide (stock solution at a concentration of 10 mM        in H₂O).    -   (5) Following peptide addition, the cells were incubated 0 to 24        hours (e.g. 30 min, 1, 6 or 24 hours) at 37° C., CO₂ 5% and 100%        relative humidity.

Acidic Wash and Cellular Extracts:

-   -   (6) The extracts were cooled on ice.

Suspension Cells (or Cells, which Don Attach Well to the Dish):

-   -   Transfer the cells in «Falcon 15 ml». To recover the maximum of        cells, wash the dish with 1 ml of PBS.    -   Harvest the cells 2 min at 2400 rpm max.    -   Suspend the cells in 1 ml cold PBS.    -   Transfer the cells into a coated “Eppendorf tube” (coated with 1        ml of poly D-Lys for 4 hours and washed twice with 1 ml PBS).    -   Wash three times with 1 ml of cold acidic wash buffer and        centrifuge 2 min at 2400 rpm max. Beware of the spreading of the        cells in the “eppendorf”.    -   Wash twice with 1 ml cold PBS to neutralize.    -   Add 50 μl of lysis RIPA Buffer.    -   Incubate 30 min-1 h on ice with agitation.

Adherent Cells:

-   -   Wash three times with 3 ml, 1 ml or 200 μl (for 6, 24 or 96 well        plates, respectively) of cold acidic wash buffer. Beware of the        cells who detach from the dish.    -   Wash twice with 1 ml cold PBS (for 6, 24 or 96 well plates,        respectively) to neutralize.    -   Add 50 μl of lysis RIPA buffer.    -   Incubate 30 min-1 h on ice with agitation.    -   Scrap the cells with a cold scrapper. The 24 and 96 well plates        were directly centrifuged at 4000 rpm at 4° for 15 mm to remove        the cellular debris. Then the supernatants (100 or 0.50 ml        respectively for the 24 or 96 well plates) were directly        transferred in a dark 96 well plated. The plates were read by a        fluorescence plate reader (Fusion Alpha, Perkin Elmer).    -   Transfer the lysate in a coated “eppendorf” (coated with 1 ml of        poly D-Lys for 4 hours and wash twice with 1 ml PBS).    -   The lysed cells were then centrifuged 30 min at 10000 g at 4° C.        to remove the cellular debris.    -   Remove the supernatant and store it at −80° C. in a coated        “Eppendorf tube” (coated with 1 ml of poly D-Lys for 4 hours and        washed twice with 1 ml PBS).

Analysis of Peptide Internalization with a Fluorescence Plate Reader:

-   -   (7) The content of each protein extract was determined by a        standard BCA assay (Kit No 23225, Pierce), following the        instructions of the manufacturer.    -   (8) The relative fluorescence of each sample is determined after        reading 10 μl of each sample in a fluorescence plate reader        (Fusion Alpha, Perkin Elmer), background subtraction and        normalization by protein concentration.

3.4. Internalization Experiments and Analysis

The time dependant internalization (uptake) of FITC-labeled TAT derivedtransporter constructs into cells of the HL-60 cell line was carried outwith materials and methods as described above.

Briefly, HL-60 cells were incubated 30 min, 1, 6 or 24 hours with 101Mof the TAT-derivative transporters. The cells were then washed twicewith an acidic buffer (0.2 M Glycin, 0.15 M NaCl, pH 3.0) and twice withPBS. Cells were broken by the addition of RIPA lysis buffer. Therelative amount of internalized peptide was then determined by readingthe fluorescence intensity (Fusion Alpha plate reader; PerkinElmer) ofeach extract followed by background substraction and protein contentnormalization. The r3-L-TAT transporter construct showed aninternalization capability as effective as the D-TAT transporterconstruct. The r3-L-TATi transporter construct, which internalized in atime dependent manner, as both previous transporters, seems to be lessefficient but still suitable, whereas L-TAT doesn't accumulate over aperiod of 24 hours (see FIG. 5).

Furthermore, a confocal microscopy was carried out with cells treatedwith fluorescently labeled TAT derived transporter constructs asdescribed above. The dissociated cortical primary neurons from P2Sprague Dawley rats were cultured 12 days in neurobasal medium beforeexposure 24 hours to 500 nM of the FITC-labeled TAT derivativetransporters. The cells were washed five times with PBS on ice and thenmounted in fluorsave mounting medium without prior fixation.Acquisitions were performed on LSM510 metaconfocal microscope (Zeiss).Images were processed with LSM510 software and mounted using Adobephotoshop. Visualization by confocal microscopy of labeling with 500 nMFITC-transporters (A: green). Nuclei were stained by Hoechst (B: blue).The r3-L-TAT as well as the D-TAT and the r3-L-TATi transporterconstructs were internalized into the cytoplasm of the non stressedneurons (C: Merge panel). However, after 24 hours incubation, the L-TATtransporter was not present anymore (see FIG. 6).

3.5. Further Internalization Experiments and Analysis

The time dependant internalization (uptake) of FITC-labeled TAT derivedtransporter constructs into cells of the HL-60 cell line was furthermorecarried out with materials and methods as described above usingsequences of the 96-FITC-labeled D-TAT derivative transporters. Thesesequences are listed below in Table 6.

TABLE 6 peptide No: SEQ abbreviation ID in FIGS. 13 NO: and 14 20r3-L-TAT H2N dR K K R dR Q R R dR CONH2 26 1 H2N dR A K R dR Q R R dRCONH2 27 2 H2N dR K A R dR Q R R dR CONH2 28 3 H2N dR K K A dR Q R R dRCONH2 29 4 H2N dR K K R dR A R R dR CONH2 30 5 H2N dR K K R dR Q A R dRCONH2 31 6 H2N dR K K R dR Q R A dR CONH2 32 7 H2N dR D K R dR Q R R dRCONH2 33 8 H2N dR K D R dR Q R R dR CONH2 34 9 H2N dR K K D dR Q R R dRCONH2 35 10 H2N dR K K R dR D R R dR CONH2 36 11 H2N dR K K R dR Q D RdR CONH2 37 12 H2N dR K K R dR Q R D dR CONH2 38 13 H2N dR E K R dR Q RR dR CONH2 39 14 H2N dR K E R dR Q R R dR CONH2 40 15 H2N dR K K E dR QR R dR CONH2 41 16 H2N dR K K R dR E R R dR CONH2 42 17 H2N dR K K R dRQ E R dR CONH2 43 18 H2N dR K K R dR Q R E dR CONH2 44 19 H2N dR F K RdR Q R R dR CONH2 45 20 H2N dR K F R dR Q R R dR CONH2 46 21 H2N dR K KF dR Q R R dR CONH2 47 22 H2N dR K K R dR F R R dR CONH2 48 23 H2N dR KK R dR Q F R dR CONH2 49 24 H2N dR K K R dR Q R F dR CONH2 50 25 H2N dRR K R dR Q R R dR CONH2 51 26 H2N dR K R R dR Q R R dR CONH2 52 27 H2NdR K K K dR Q R R dR CONH2 53 28 H2N dR K K R dR R R R dR CONH2 54 29H2N dR K K R dR Q K R dR CONH2 55 30 H2N dR K K R dR Q R K dR CONH2 5631 H2N dR H K R dR Q R R dR CONH2 57 32 H2N dR K H R dR Q R R dR CONH258 33 H2N dR K K H dR Q R R dR CONH2 59 34 H2N dR K K R dR H R R dRCONH2 60 35 H2N dR K K R dR Q H R dR CONH2 61 36 H2N dR K K R dR Q R HdR CONH2 62 37 H2N dR I K R dR Q R R dR CONH2 63 38 H2N dR K I R dR Q RR dR CONH2 64 39 H2N dR K K I dR Q R R dR CONH2 65 40 H2N dR K K R dR IR R dR CONH2 66 41 H2N dR K K R dR Q I R dR CONH2 67 42 H2N dR K K R dRQ R I dR CONH2 68 43 H2N dR L K R dR Q R R dR CONH2 251 44 H2N dR dR dRdQ dR dR dK dK dR CONH2 (D-TAT) 21 45 H2N dR R R Q dR R K K dR CONH2(r3-L-TATi) 20 46 H2N dR K K R dR Q R R dR CONH2 (r3-L-TAT) 18 47 H2N RK K R R Q R R R CONH2 (L-TAT) 73 48 H2N dR K K R dR Q R L dR CONH2 74 49H2N dR M K R dR Q R R dR CONH2 75 50 H2N dR K M R dR Q R R dR CONH2 7651 H2N dR K K M dR Q R R dR CONH2 77 52 H2N dR K K R dR M R R dR CONH278 53 H2N dR K K R dR Q M R dR CONH2 79 54 H2N dR K K R dR Q R M dRCONH2 80 55 H2N dR N K R dR Q R R dR CONH2 81 56 H2N dR K N R dR Q R RdR CONH2 82 57 H2N dR K K N dR Q R R dR CONH2 83 58 H2N dR K K R dR N RR dR CONH2 84 59 H2N dR K K R dR Q N R dR CONH2 85 60 H2N dR K K R dR QR N dR CONH2 86 61 H2N dR Q K R dR Q R R dR CONH2 87 62 H2N dR K Q R dRQ R R dR CONH2 88 63 H2N dR K K Q dR Q R R dR CONH2 89 64 H2N dR K K RdR K R R dR CONH2 90 65 H2N dR K K R dR Q Q R dR CONH2 91 66 H2N dR K KR dR Q R Q dR CONH2 92 67 H2N dR S K R dR Q R R dR CONH2 93 68 H2N dR KS R dR Q R R dR CONH2 94 69 H2N dR K K S dR Q R R dR CONH2 95 70 H2N dRK K R dR S R R dR CONH2 96 71 H2N dR K K R dR Q S R dR CONH2 97 72 H2NdR K K R dR Q R S dR CONH2 98 73 H2N dR T K R dR Q R R dR CONH2 99 74H2N dR K T R dR Q R R dR CONH2 100 75 H2N dR K K T dR Q R R dR CONH2 10176 H2N dR K K R dR T R R dR CONH2 102 77 H2N dR K K R dR Q T R dR CONH2103 78 H2N dR K K R dR Q R T dR CONH2 104 79 H2N dR V K R dR Q R R dRCONH2 105 80 H2N dR K V R dR Q R R dR CONH2 106 81 H2N dR K K V dR Q R RdR CONH2 107 82 H2N dR K K R dR V R R dR CONH2 108 83 H2N dR K K R dR QV R dR CONH2 109 84 H2N dR K K R dR Q R V dR CONH2 110 85 H2N dR W K RdR Q R R dR CONH2 111 86 H2N dR K W R dR Q R R dR CONH2 112 87 H2N dR KK W dR Q R R dR CONH2 113 88 H2N dR K K R dR W R R dR CONH2 114 89 H2NdR K K R dR Q W R dR CONH2 115 90 H2N dR K K R dR Q R W dR CONH2 116 91H2N dR Y K R dR Q R R dR CONH2 117 92 H2N dR K Y R dR Q R R dR CONH2 11893 H2N dR K K Y dR Q R R dR CONH2 119 94 H2N dR K K R dR Y R R dR CONH2120 95 H2N dR K K R dR Q Y R dR CONH2 121 96 H2N dR K K R dR Q R Y dRCONH2

In the above table D amino acids are indicated by a small “d” prior tothe respective amino acid residue (dB=D-Arg).

For a few sequences synthesis failed in the first approach unfortunatelydue to technical reasons. These sequences are abbreviated in FIG. 13 as1, 2, 3, 4, 5, 6, 7, 8, 43, 52, 53, 54, 55, 56, 57, 85, 86, 87, 88, 89,and 90. However, the remaining sequences were used in theinternalization experiments.

The results are shown in FIGS. 13 and 14.

As can be seen in FIG. 13, after 24 hours incubation, all transporterswith the consensus sequence rXXXrXXXr (SEQ ID NO: 752) (see above for aselection of possible sequences) showed a higher internalizationcapability than the L-TAT transporter. Hela cells were incubated 24hours in 96 well plate with 10 mM of the r3-L-TAT-derived transporters.The cells were then washed twice with an acidic buffer (0.2M Glycin,0.15 M NaCl, pH 3.0) and twice with PBS. Cells were broken by theaddition of RIPA lysis buffer. The relative amount of internalizedpeptide was then determined by reading the fluorescence intensity(Fusion Alpha plate reader; PerkinElmer) of each extract followed bybackground subtraction

As can be seen in FIG. 14, one positions appears to be critical forhighest transporter activity and for improved kinetics of transportactivity: Y in position 2 (peptide No. 91 corresponding to SEQ ID NO:116). Briefly, Hela cells were incubated 2, 6 or 24 hours in 24 wellplate with increasing dose of the r3-L-TAT-derivative transporters (0,500 nM, 1 mM or 10 mM). The cells were then washed twice with an acidicbuffer (0.2M Glycin, 0.15 M NaCl, pH 3.0) and twice with PBS. Cells werebroken by the addition of RIPA lysis buffer. The relative amount ofinternalized peptide was then determined by reading the fluorescenceintensity (Fusion Alpha plate reader; PerkinElmer) of each extractfollowed by background substraction.

The conclusion of this experiment is as follows:

-   -   After 24 hours incubation, all transporters with the consensus        sequence rXXXrXXXr (SEQ ID NO: 252) (see Table 1 for a selection        of possible sequences) showed a higher internalization        capability than the L-TAT transporter (FIG. 13). Those results        fully validate the consensus sequence rXXXrXXXr (SEQ ID NO:        252).    -   One position is critical for highest transporter activity and        (FIG. 13): Y in position 2 (sequence 91 corresponding to SEQ ID        NO: 116).    -   One position is critical for improved kinetics of transport        activity (FIG. 14): Y in position 2 (sequence 91 corresponding        to SEQ ID NO: 116).

Accordingly, such TAT derived sequences as shown in Table 1 arepreferred, which exhibit an Y in position 2, particularly when thesequence according to generic formula (I) exhibits 9 aa and has theconsensus sequence rXXXrXXXr (SEQ ID NO: 252).

4. Determination of Intracellular Concentration of Specific TransporterConstructs Subsequent to Uptake (Internalization) of these Peptides intoU937 Cells

According to a further experiment, the concentration of specifictransporter constructs subsequent to uptake (internalization) of thesepeptides into U937 cells were determined. The experiments were carriedout using the sequences RKKRRQRRR (L-TAT), rrrqrrkkr (D-TAT), rKKRrQRRr(r3-L-TAT) and rYKRrQRRr (XG-91), each in a concentration of 10 μM each.

10 μM 2 h 4 h 6 h 24 h RKKRRQRRR (L-TAT) 1.20 1.38 1.07  0.5(SEQ ID NO: 18) rrrqrrkkr (D-TAT) 2.00 2.24 3.55 17.3 (SEQ ID NO: 251)rKKRrQRRr (r3-L-TAT) 2.34 3.16 3.56 11.2 (SEQ ID NO: 20)rYKRrQRRr (XG-91, 3.16 4.27 4.68 50   sequence 91 correspondingto SEQ ID NO: 116)

Surprisingly, the accumulation of rYKRrQRRr (XG-91, sequence 91corresponding to SEQ ID NO: 116), shows an extremely accumulation in thecell, which is even significantly higher than the concentration of thetransporter construct in the medium or the average concentration ofabout 20 μNI which was expected for D-TAT construct. This underlines theimportance of transporter constructs according to generic formula (I),particular of transporter constructs which comprise a TAT derivedsequence as shown in Table 1, which exhibits an Y in position 2, andpreferably has 9 aa and the consensus sequence rXXXrXXXr (SEQ ID NO:252).

5. Uptake (Internalization) of Peptides into Cells and Measurement ofPeptide Internalization in Cell Lines HepG2 (Hepatocarcinoma), HCT-116(Tumoral Colon), U937 (Lymphoma), in WBC Cell Lines (White Blood CellLines) and Non-WBC Cell Lines

In these experiments, the internalization (uptake) capacity of ofFITC-labeled TAT derived transporter constructs in vitro was evaluatedwith a fluorescence plate reader in further cell lines HepG2(hepatocarcinoma), HCT-116 (tumoral colon), U937 (Lymphoma), in WBC celllines (white blood cell lines) and non-WBC cell lines.

Test Samples and Conditions Used in the Experiments

The constructs and conditions used in this experiment were as describedabove for experiment 3 with following amendments and cell lines:

a) Uptake (Internalization) of FITC-Labeled TAT Derived TransporterConstructs In Vitro (10 HepG2 Hepatocarcinoma, HCT-116 Tumoral Colon, 24h)

The constructs used were different TAT derived transporter constructstermed D-TAT and r3-TATi (also termed r3-L-TATi), D-TAT, each having alength of 9 amino acids but a different D-/L-pattern, and the constructsr₆R₅ and DAK, wherein the constructs additionally have been labeled withbeta-Alanine at their N-terminus. The results are shown in FIG. 7. Ascan be seen in FIG. 7, the uptake was most efficient for constructsD-TAT and r₆R₃, followed by r₃-L-TATi.

b) Uptake (Internalization) of FITC-Labeled TAT Derived TransporterConstructs In Vitro (10 μM, U937, Lymphoma, 24 h).

The constructs used were four different TAT derived transporterconstructs (termed L-TAT, r3-TAT (also termed r3-L-Tat), r3-TATi (alsotermed r3-L-TATi), and D-TAT), each having a length of 9 amino acids buta different D-/L-pattern. Additionally, the construct DAK was used forcomparison and a control sample, containing no peptide. The results areshown in FIG. 8. As can be seen in FIG. 8, the uptake of r3-TAT, r3-TATiand D-TAT transporter constructs into the cells was most efficient,wherein L-TAT showed a significantly lower uptake into the cells.

c) HSPG Dependency of Uptake (Internalization) of the D-TAT TransporterConstruct

An experiment was carried out to see, whether the uptake(internalization) of the D-TAT transporter construct is HSPG-dependent.As found, the uptake (internalization) of the D-TAT transporterconstruct is HSPG-dependent at a concentration of 500 nm over 24 hoursin U937 cells, Lymphoma (see also FIG. 9). The construct used for theexperiment was D-TAT, having a length of 9 amino acids and being labeledwith FITC and at its N-terminus with beta-Alanine.

d) Exit of the FITC-Labeled TAT Derived Transporter Constructs in U937Cells (lymphoma)

A further experiment was carried out to see, whether the FITC-labeledTAT derived transporter constructs exit U937 cells. As a result, an exitis not observed in U937 cells at 500 nM FITC-D-TAT (see FIG. 10). Theconstruct used for the experiment was D-TAT (SEQ ID NO: 251), having alength of 9 amino acids and being labeled with FITC and at itsN-terminus with beta-Alanine.

Furthermore, it could be seen, that an exit of the FITC-labeled TATderived transporter constructs is observed at 10 μM FITC-D-TAT, and isHSPG-dependent in U937 cells (lymphoma) (see Figure ii). The constructused for the experiment was again D-TAT (SEQ ID NO: 251) as above.

e) Uptake (Internalization) and an Exit of the FITC-Labeled TAT DerivedTransporter Constructs at 10 μM FITC-D-TAT in Non WBC-Lines (White BloodCells Lines)

In a further experiment an uptake (internalization) and an exit of theFITC-labeled TAT derived transporter constructs are observed at 10 μMFITC-D-TAT in non WBC-lines (white blood cells lines) (see FIG. 12). Theconstruct used for the experiment was D-TAT (SEQ ID NO: 251), having alength of 9 amino acids and being labeled with FITC and at itsN-terminus with beta-Alanine.

f) Conclusions

As a conclusion of the above uptake (internalization) experiments and ascan be seen in the above Figures, uptake (internalization) ofFITC-labeled TAT derived transporter constructs containing orexclusively composed of D-amino acids is linear over several hrs invitro. Furthermore, at 24 hrs, the uptake (internalization) of theseFITC-labeled TAT derived transporter constructs in vitro reaches 50-100fold higher intracellular concentrations than L-TAT. Additionally, theuptake (internalization) of FITC-labeled TAT derived transporterconstructs containing or exclusively composed of D-amino acids byWBC-lines (white blood cells lines) is 10-50 fold more efficient than bynon-WBC-lines in vitro. For all these experiments, an exit was shown tobe efficient at high intracellular concentration, but is not observed atlow concentrations in WBCs

6. Synthesis of Cytotoxic Transporter Cargo Conjugate MoleculeD-Tat-Cisplatin, r3-L-TAT-Cisplatin and r3-L-TATi-Cisplatin

6.1Peptide Synthesis

The peptide sequence of D-TAT (rrrqrrkkr; SEQ ID NO: 251), r3-L-TAT(rKKRrQRRr; SEQ ID NO: 20) and r3-L-TATi (rRRQrRKKr; SEQ ID NO: 21)including an additional methionine is synthesized manually on 0.4 mmolFmoc-Amide-AM resin by using Fmoc chemistry. The peptide is then cleavedfrom the resin with TFA, filtered under a reduced pressure, precipitatedwith cold ether, and dried. The crude peptide is purified bySemi-preparative HPLC and characterized by ESI-MS.

6.2Alkylation of Peptide to Cisplatin

5.0 μmol of Cisplatin (1.5 mg in 3.0 ml Sodium Chloride buffer, pH 5.0)are dissolved in 2.0 ml of 10 mM Na₂HPO₄ buffer (pH 7.4), and pH valueof the solution is 7.0. 5.0 μmol of D-TAT-Methionine peptide (or ofr3-L-TAT-Methionine peptide or of r3-L-TATi-Methionine peptide) isprepared in 10 mM Na₂HPO₄ buffer (pH 7.4) and pH value of the solutionis 6.0. Then the alkylation is started by mixing two solutions at roomtemperature in dark (pH value of the mixture is 7.0). After 0 h, 1 h, 3h and 24 h, the product is analysed by analytic RP-HPLC, andcharacterized by ESI-MS. The expected peak solution is finally purifiedby Semi-preparative RP-HPLC and lyophilized.

7. Synthesis of Cytotoxic Transporter Cargo Conjugate MoleculeD-Tat-Oxaliplatin, r3-L-TAT-Oxaliplatin and r3-L-TATi-Oxaliplatin

7.1. Peptide (D-Tat-Methionine, r3-L-TAT-Methionine andr3-L-TATi-Methionine) Synthesis

The peptide sequence of D-TAT (rrrqrrkkr, SEQ ID NO: 251), r3-L-TAT(rKKRrQRRr; SEQ ID NO: 20) and r3-L-TATi (rRRQrRKKr; SEQ ID NO: 21) issynthesized manually on 0.23 mmol Fmoc-Rink Amide resin by using Fmocchemistry. Thus, each amino acid from C-terminal Gly to N-terminal 1-Met(L-Form) is sequentially attached to the resin with with a cycle ofFmoc-deprotection (20% piperidine in DMF) and amino acid coupling(HBTU/HOBt/DIEA in DMF activation). The peptide is cleaved from theresin with TFA (2h in the presence of 2.5% dH₂O, 0.5% EDT and 2.0% TIS),filtred at atmospheric pressure, volume reduced by N₂ bubbling,precipitated with cold ether and air-dried. The crude peptide ispurified by semi-preparative RP-HPLC and characterized by ESI-MS.

7.2. Alkylation of Peptide to Oxaliplatin

10 μmol Oxaliplatin, formulated as Eloxatin® (Oxaliplatinum 4.0 mg,lactosum monohydricum 36.0 mg) in 5.0 ml 10 mM Na₂HPO₄ buffer (pH 7.4).10 μmol of D-Tat-Methionine peptide (or of r3-L-TAT-Methionine peptideor of r3-L-TATi-Methionine peptide) is prepared in dH₂O 5.0 ml.Alkylation is started by mixing the two solutions at room temperature.Reaction is then left at 37° C. and monitored by analytical RP-HPLC at214 and 280 nm over 24h, target peak is characterized by ESI-MS andpurified by semi-preparative RP-HPLC followed by lyophilization.

7.3. Test Conditions

Effects of a treatment with increasing concentrations of a conjugatemolecule of the invention (D-Tat-oxaliplatin, r3-L-TAT-oxaliplatin, orr3-L-TATi-oxaliplatin) on the survival of MCF-7 (human breastadenocarcinoma cell line) and SiHa (human cervix squamous carcinoma cellline) are determined. The effects of D-Tat-oxaliplatin,r3-L-TAT-oxaliplatin, or r3-L-TATi-oxaliplatin is compared to theconjugate L-Tat-oxaliplatin and to two unconjugated anti-cancer drugs(Oxaliplatin and Cisplatin). Cells of each cell line (10,000 cells perwell) are plated into 96 well plates (200 μl total volume of MEMsupplemented with 10% FBS, 1% L-glutamine, 1% Na-pyruvate, 1%non-essential amino acids for MCF-7 and of MEM/Earle's supplemented with10% FBS, 1% Na-pyruvate, 1% non-essential amino acids for SiHa cells). 6to 10 different concentrations for each test substance are tested. Thecontrol cells are non-treated. Cells are incubated at 37° C. for 24hbefore treatment with the test substance. Each experiment is carried intriplicate. Cell incubation after treatment is performed for 96 hours at37° C. The effects of the test molecules on the survival of these celllines (in vitro cytotoxic activity) is measured by the MTT assay. 20 μlof a 5 mg/ml 0.22 μm filtered Thiazolyl Blue Tetrazolium Bromidesolution (MTT, Sigma, Ref. No. 88415) in Phosphate Buffered saline (PBS,CHUV) are added to each well and the plate is incubated for 4 hours at37° C. The supernatant is removed and formazan crystals are dissolvedwith DMSO (200 μl per well). Absorbancy (OD) is measured in a microplatereader at 595 nm (Expert Plus Reader, Asys Hitech). The IC₅₀(concentration of the drug inhibiting 50% of the cell growth) for thetest substances is calculated using Prism software.

8. Synthesis of Cytotoxic Transporter Cargo Conjugate MoleculeD-Tat-Chlorambucil, r3-L-TAT-Chlorambucil and r3-L-TATi-Chlorambucil

8.1 Conjugate Molecule (D-Tat-Chlorambucil, r3-L-TAT-Chlorambucil andr3-L-TATi-Chlorambucil) synthesis

The D-TAT (rrrqrrkkr; SEQ ID NO: 251), r3-L-TAT (rKKRrQRRr; SEQ ID NO:20) or r3-L-TATi (rRRQrRKKr; SEQ ID NO: 21) peptide sequence issynthesized manually on 0.23 mmol Fmoc-Rink Amide resin by using Fmocchemistry. Thus, each amino acid from C-terminal Gly to N-terminal 1-□A(L-form) is sequentially attached to the resin with with a cycle ofFmoc-deprotection (20% piperidine in DMF) and amino acid coupling(HBTU/HOBt/DIEA in DMF activation).

Following Fmoc-deprotection (20% piperidine in DMF) of N-terminal 1-EA,coupling of chlorambucil is achieved using standard amino acid couplingconditions (HBTU/HOBt/DIEA in DMF activation). The conjugate molecule iscleaved from the resin with TFA (70 min in the presence of 3% dH₂O and3% TIS), filtred at atmospheric pressure, volume reduced by N₂ bubbling,precipitated with cold ether and air-dried. The crude conjugate moleculeis purified by semi-preparative RP-HPLC, characterized by ESI-MSfollowed by lyophilization.

8.1 Comparative Studies

Effects of a treatment with increasing concentrations ofD-Tat-chlorambucil, r3-L-TAT-chlorambucil, or r3-L-TATi-chlorambucil onthe survival of MCF-7 (human breast adenocarcinoma cell line) and SiHa(human cervix squamous carcinoma cell line) is determined. The effectsof D-Tat-chlorambucil, r3-L-TAT-chlorambucil, or r3-L-TATi-chlorambucilis furthermore compared to the conjugate L-Tat-chlorambucil and to two uchlorambucil conjugated anti-cancer drugs (Chlorambucil and Cisplatin).Cells of each cell line (10,000 cells per well) are plated into 96 wellplates (200 μl total volume of MEM supplemented with 10% FBS, 1%L-glutamine, 1% Na-pyruvate, 1% non-essential amino acids for MCF-7 andof MEM/Earle's supplemented with 10% FBS, 1% Na-pyruvate, 1%non-essential amino acids for SiHa cells). 6 to 10 differentconcentrations for each test substance are tested. The control cells arenon-treated. Cells are incubated at 37° C. for 24 h before treatmentwith the test substance. Each experiment is carried in triplicate. Cellincubation after treatment is performed for 96 hours at 37° C. Theeffects of the test molecules on the survival of these cell lines (invitro cytotoxic activity) is measured by the MTT assay. 20 μl of a 5mg/ml 0.22 lam filtered Thiazolyl Blue Tetrazolium Bromide solution(MTT, Sigma, Ref. No. 88415) in Phosphate Buffered saline (PBS, CHUV)are added to each well and the plate is incubated for 4 hours at 37° C.The supernatant is removed and formazan crystals are dissolved with DMSO(200 μl per well). Absorbancy (OD) is measured in a microplate reader at595 nm (Expert Plus Reader, Asys Hitech). The IC₅₀ (concentration of thedrug inhibiting 50% of the cell growth) for the test substances iscalculated using Prism software.

9. Synthesis of Cytotoxic Transporter Cargo Conjugate MoleculeD-Tat-Doxorubicine, r3-L-TAT-Doxorubicine and r3-L-TATi-Doxorubicine

9.1. Conjugate Molecule (D-Tat-Doxorubicine, r3-L-TAT-Doxorubicine andr3-L-TATi-Doxorubicine) Synthesis

The D-TAT (rrrqrrkkr; SEQ ID NO: 251), r3-L-TAT (rKKRrQRRr; SEQ ID NO:20) and r3-L-TATi (rRRQrRKKr; SEQ ID NO: 21) peptide sequence issynthesized manually on 0.23 mmol Fmoc-Rink Amide resin by using Fmocchemistry. Thus, each amino acid from C-terminal Gly to N-terminal I-E(L-form) is sequentially attached to the resin with with a cycle ofFmoc-deprotection (20% piperidine in DMF) and amino acid coupling(HBTU/HOBt/DIEA in DMF activation).

Following Fmoc-deprotection (20% piperidine in DMF) of N-terminal l-E,acetylation (acetic anhydride, DIEA in DMF activation) is done. Removalof the Odmab side-chain protecting group is performed using 2% hydrazinemonohydrate in DMF. Coupling of chlorambucil formulated as Adriblastin®(Doxorubicinie.HCl 18%, NaCl 82% lyophilized) is achieved via OBt ester(DIPCDI/HOBt/DIEA in DCM/DMF activation).

The conjugate molecule is cleaved from the resin with TFA (2 h in thepresence of 1.7% dH₂O and 1.7% TIS), filtred at atmospheric pressure,volume reduced by N₂ bubbling, precipitated with cold ether andair-dried. The crude conjugate molecule is purified by semi-preparativeRP-HPLC, characterized by ESI-MS followed by lyophilization.

10. Synthesis of Cytotoxic Transporter Cargo Conjugate MoleculeD-Tat-Saquinavir, r3-L-TAT-Saquinavir and r3-L-TATi-Saquinavir

10.1. Peptide (D-TAT-D-Cysteine r3-L-TAT-D-Cysteine andr3-L-TATi-D-Cysteine) Synthesis

The D-TAT (rrrqrrkkr; SEQ ID NO: 251), r3-L-TAT (rKKRrQRRr; SEQ ID NO:20) and r3-L-TATi (rRRQrRKKr; SEQ ID NO: 21) peptide sequence issynthesized manually on 0.40 mmol Fmoc-Rink Amide resin by using Fmocchemistry. Thus, each amino acid from C-terminal D-Arg to N-terminalD-Cys is sequentially attached to the resin with with a cycle ofFmoc-deprotection (20% piperidine in DMF) and amino acid coupling(TBTU/HOBt/DIEA in DMF activation). The peptide is cleaved from theresin with TFA, pre-incubated on ice (5h in the presence of 2.5% dH₂O,2.5% EDT and 1.0% TIS), filtred at reduced pressure, precipitated withcold ether and vacuum dried. The crude peptide is purified bysemi-preparative RP-HPLC and characterized by ESI-MS.

10.2. Preparation of Saquinavir Active Ester

375 μmol Boc-Gly-OH is dissolved in anhydrous DCM at room temperature,and to this is added 265 μmol DMAP, 37.51.1 mol DIPCI and 110 μmolSaquinavir, formulated as Invirase® (lactose, excipiens pro compressoobducto) at 0° C. The reaction mixture is allowed to warm to roomtemperature and stirred overnight. The product is ished with 0.1N HCl,dried over MgSO₄, and evaporated under reduced pressure to yield thesolid product SQV-Gly(Boc). The Boc protecting group is removed byincubating SQV-Gly(Boc) ester for 3 h in a mixture of CH₂Cl₂ and TFA(50:50). The product is recristallized from cold ether and dried undervacuum overnight. 47 μmol SQV-Gly ester is dissolved in 3 ml anhydrousDMSO at room temperature, and to this is added 94 μmol SPDP. Thereaction mixture pH is adjusted to 8.0 under constant stirring at roomtemperature. The reaction is left for 3h under constant stirring. Thecrude product SQV-Gly-COCH2CH2-SS-pyridyl is purified bysemi-preparative RP-HPLC and characterized by ESI-MS.

10.3. Conjugation of Peptide D-TAT (Rrrqrrkkr; SEQ ID NO: 251), r3-L-TAT(rKKRrQRRr; SEQ ID NO: 20) or r3-L-TATi (rRRQrRKKr; SEQ ID NO:21)-D-Cysteine to Saquinavir

27 μmol SQV-Gly-COCH2CH2-SS-pyridyl is dissolved in 0.5 ml PBS buffer pH7.5 at room temperature, and to this is added 54 μmol D-TAT (rrrqrrkkr,SEQ ID NO: 251), r3-L-TAT (rKKRrQRRr; SEQ ID NO: 20) or r3-L-TATi(rRRQrRKKr; SEQ ID NO: 21)-D-Cysteine in 0.5 ml PBS buffer pH 7.5. Thereaction is left at room temperature for 3 h under constant stirring.The crude conjugate D-TAT (rrrqrrkkr, SEQ ID NO: 251)-Saquinavir,r3-L-TAT (rKKRrQRRr, SEQ ID NO: 20)-Saquinavir and r3-L-TATi (rRRQrRKKr;SEQ ID NO: 21)-Saquinavir is purified by semi-preparative RP-HPLC andcharacterized by ESI-MS.

11. Preparation of Transporter Cargo Conjugate Molecules Comprising JNKInhibitor Sequences as Cargo Moieties 11.1. Identification of JNKInhibitor Sequences

Amino acid sequences important for efficient interaction with JNK areidentified by sequence alignments between known JBDs, e.g. between theJBDs of IB1, IB2, c-Jun and ATF2 defined a weakly conserved 8 amino acidsequence. Using this alignment JNK inhibitor sequences could beidentified leading to a number of suitable JNK inhibitor sequencesdefined herein as SEQ ID NOs: 137 to 220.

11.2. Preparation of Transporter Cargo Conjugate Molecules ComprisingJNK Inhibitor Sequences

Transporter cargo conjugate molecules comprising the above JNK Inhibitorsequences are synthesized by covalently linking the C-terminal end ofJNK inhibitor sequences as defined herein to an N-terminal transporterconstruct according to generic formula (I) as defined above. Thetransporter construct according to generic formula (I) as defined aboveis prepared using classical Fmock synthesis and further analysed by MassSpectrometry. The components are finally purified by HPLC. Linkage maybe carried out using a linker consisting of two proline residues. Thislinker can be used to allow for maximal flexibility and prevent unwantedsecondary structural changes.

11.3 Inhibition of Cell Death

Effects of the on JNK biological activities are studied. The constructis linked N-terminally to the Green Fluorescent Protein and the effectof this construct on pancreatic β-cell apoptosis induced by IL1 isevaluated. This mode of apoptosis is previously shown to be blocked bytransfection with JBD₁₋₂₈₀ whereas specific inhibitors of ERK1/2 or p38did not protect.

Insulin producing □TC-3 cells are cultured in RPMI 1640 mediumsupplemented with 10% Fetal Calf Serum, 100 μg/mL Streptomycin, 100units/mL Penicillin and 2 mM Glutamine. Insulin producing □TC-3 cellsare transfected with the inventive tansporter cargo conjugate moleculesand IL-1□ (10 ng/mL) is added to the cell culture medium. The number ofapoptotic cells is counted at 48 hours after the addition of IL-1□ usingan inverted fluorescence microscope. Apoptotic cells are discriminatedfrom normal cells by the characteristic “blebbing out” of the cytoplasmand are counted after two days.

12. Cellular Import of Inventive Transporter Cargo Conjugate MoleculesComprising TAT Derived Transporter Constructs According to SEQ ID NOs: 8to 136 and JNK1 or IB1 Derived Cargo Peptides According to any of SEQ IDNOs: 137 to 220

The ability of the transporter cargo conjugate molecules comprising TATderived transporter constructs according to any of SEQ ID NOs: 8 to 136and JNK1 or IB1 derived cargo peptides according to any of SEQ ID NOs:137 to 220 to enter cells is evaluated. Inventive transporter constructsand inventive transporter cargo conjugate molecules are labeled byN-terminal addition of a glycine residue conjugated to fluorescein.These labeled peptides (1 μM) are added to □TC-3 cell cultures, whichare maintained as described in Example 11. At predetermined times cellsare fished with PBS and fixed for five minutes in ice-coldmethanol-acetone (1:1) before being examined under a fluorescencemicroscope. Fluorescein-labeled BSA (1 μM, 12 moles/mole BSA) is used asa control.

Fluorescent signals from these transporter constructs and inventivetransporter cargo conjugate molecules are determined.

13. In Vitro Inhibition of c-JUN, ATF2 and Elk1 Phosphorylation byInventive Transporter Cargo Conjugate Molecules Comprising TAT DerivedTransporter Constructs According to any of SEQ ID NOs: 8 to 136 and JNK1or IB1 Derived Cargo Peptides According to any of SEQ ID NOs: 137 to 220

The effects of the inventive transporter cargo conjugate moleculescomprising TAT derived transporter constructs according to any of SEQ IDNOs: 8 to 136 and JNK1 or IB1 derived cargo peptides according to any ofSEQ ID NOs: 137 to 220 on JNKs-mediated phosphorylation of their targettranscription factors are investigated in vitro. Recombinant and nonactivated JNK1, JNK2 and JNK3 are produced using a TRANSCRIPTION ANDTRANSLATION rabbit reticulocyte lysate kit (Promega) and used in solidphase kinase assays with c-Jun, ATF2 and Elk1, either alone or fused toglutathione-S-transferase (GST), as substrates. Dose response studiesare performed wherein inventive transporter cargo conjugate moleculescomprising TAT derived transporter constructs according to any of SEQ IDNOs: 8 to 136 and JNK1 or IB1 derived cargo peptides according to any ofSEQ ID NOs: 137 to 220 are mixed with the recombinant JNK1, JNK2, orJNK3 kinases in reaction buffer (20 mM Tris-acetate, 1 mM EGTA, 10 mMp-nitrophenyl-phosphate (pNPP), 5 mM sodium pyrophosphate, 10 mMp-glycerophosphate, 1 mM dithiothreitol) for 20 minutes. The kinasereactions are then initiated by the addition of 10 mM MgCl₂ and 5 pCi³³P-□-dATP and 1 μg of either GST-Jun (aa 1-89), GST-AFT2 (aa 1-96) orGST-ELK (aa 307-428). GST-fusion proteins are purchased from Stratagene(La Jolla, Calif.).

Ten μL of glutathione-agarose beads are also added to the mixture.Reaction products are then separated by SDS-PAGE on a denaturing 10%polyacrylamide gel. Gels are dried and subsequently exposed to X-rayfilms (Kodak) and inhibition of c-Jun, ATF2 and Elk1 phosphorylation bythese inventive transporter cargo conjugate molecules comprising TATderived transporter constructs according to any of SEQ ID NOs: 8 to 136and JNK1 or IB1 derived cargo peptides according to any of SEQ ID NOs:137 to 220 is determined.

14. Inhibition of IL-1□ Induced Pancreatic β-Cell Death by InventiveTransporter Cargo Conjugate Molecules Comprising TAT Derived TransporterConstructs According to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1Derived Cargo Peptides According to any of SEQ ID NOs: 137 to 220

The effects of the inventive transporter cargo conjugate moleculescomprising TAT derived transporter constructs according to any of SEQ IDNOs: 8 to 136 and JNK1 or IB1 derived cargo peptides according to any ofSEQ ID NOs: 137 to 220 on the promotion of □-cell apoptosis elicited byIL-1 is determined. TC-3 cell cultures are incubated for 30 minutes with1 μM of inventive L-TAT-IB1(s) peptides followed by 10 ng/mL of IL-1. Asecond addition of peptide (1 μM) is performed 24 hours later. Apoptoticcells are counted after two days of incubation with IL-1 β usingpropidium iodide (red stained cell are dead cells) and Hoechst 33342(blue stained cell are cells with intact plasma membrane) nuclearstaining. Addition of the inventive TAT-IB(s) peptides inhibitedIL-1-induced apoptosis of □TC-3 cells cultured in the presence of IL-1 βfor two days.

Long term inhibition of IL-1 induced cells death is examined by treating□TC-3 cells as described above, except that incubation of the cells withthe inventive transporter cargo conjugate molecules comprising TATderived transporter constructs according to any of SEQ ID NOs: 8 to 136and JNK1 or IB1 derived cargo peptides according to any of SEQ ID NOs:137 to 220 and IL-1□□□ is sustained for 12 days. Additional peptides (1μM) are added each day and additional IL-1□□ (10 ng/mL) is added every 2days.

15. Inhibition of Irradiation Induced Pancreatic β-Cell Death byInventive Transporter Cargo Conjugate Molecules Comprising TAT DerivedTransporter Constructs According to any of SEQ ID NOs: 8 to 136 and JNK1or IB1 Derived Cargo Peptides According to any of SEQ ID NOs: 137 to 220

JNK is also activated by ionizing radiation. To determine whetherinventive transporter cargo conjugate molecules comprising TAT derivedtransporter constructs according to any of SEQ ID NOs: 8 to 136 and JNK1or IB1 derived cargo peptides according to any of SEQ ID NOs: 137 to 220would provide protection against radiation-induced JNK damage, “WiDr”cells are irradiated (30 Gy) in presence or absence of D-TAT (SEQ ID NO:251), L-TAT (SEQ ID NO: 18) and inventive transporter cargo conjugatemolecules comprising TAT derived transporter constructs according to anyof SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargo peptides accordingto any of SEQ ID NOs: 137 to 220 (1 μM added 30 minutes beforeirradiation). Control cells (CTRL) are not irradiated. Cells areanalyzed 48 hours later by means of PI and Hoechst 3342 staining, asdescribed above. N=3, SEM are indicated.

16. Radioprotection to Ionizing Radiation by Inventive Transporter CargoConjugate Molecules Comprising TAT Derived Transporter ConstructsAccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 Derived CargoPeptides According to any of SEQ ID NOs: 137 to 220

To determine the radioprotective effects of the inventive transportercargo conjugate molecules comprising TAT derived transporter constructsaccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargopeptides according to any of SEQ ID NOs: 137 to 220, C57B1/6 mice (2 to3 months old) are irradiated with a Phillips RT 250 R-ray at a dose rateof 0.74 Gy/min (17 mA, 0.5 mm Cu filter). Thirty minutes prior toirradiation, the animals are injected i.p. with inventive transportercargo conjugate molecules comprising TAT derived transporter constructsaccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargopeptides according to any of SEQ ID NOs: 137 to 220. Briefly, mice areirradiated as follows: mice are placed in small plastic boxes with thehead lying outside the box. The animals are placed on their back underthe irradiator, and their neck fixed in a small plastic tunnel tomaintain their head in a correct position. The body is protected withlead. Prior to irradiation mice are maintained on standard pellet mousechow, however post irradiation mice are fed with a semi-liquid food thatis renewed each day. The reaction of the lip mucosa is then scored by 2independent observers according to the scoring system developed byParkins et al. (Parkins et al, Radiotherapy & Oncology, 1: 165-173,1983), in which the erythema status as well as the presence of edema,desquamation and exudation is quoted. Additionally, animals are weighedbefore each recording of their erythema/edema status.

17. Suppression of JNK Transcription Factors by Inventive TransporterCargo Conjugate Molecules Comprising TAT Derived Transporter ConstructsAccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 Derived CargoPeptides According to any of SEQ ID NOs: 137 to 220

Gel retardation assays are carried out with an AP-1 doubled labeledprobe (5′-CGC TTG ATG AGT CAG CCG GAA-3′ (SEQ ID NO: 262). HeLa cellnuclear extracts that are treated or not for one hour with 5 ng/mlTNF-α, as indicated. TAT and inventive transporter cargo conjugatemolecules comprising TAT derived transporter constructs according to anyof SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargo peptides accordingto any of SEQ ID NOs: 137 to 220 are added 30 minutes before TNF-α.

18. Evaluation of the Neuroprotection Against Focal Cerebral Ischemia,in a Permanent MCAO Model—Determination of the Efficacity of theProtection at Different Doses.

Focal cerebral ischemia is induced in 12-days-old rats. Pups areanesthetized in an induction chamber with 2% isoflurane and during theoperation anaesthesia is maintained using a mask under 2% isoflurane.MCAO is induced by electrocoagulating a main branch of the middlecerebral artery (MCA). Rats are placed on the right side, and an obliquedermal incision is made between the ear and eye. After excision of thetemporal muscle, the cranial bone is removed from the frontal suture toa level below the zygomatic arch. The left MCA, exposed just after itsapparition over the rhinal fissure, is permanently electrocoagulated atthe inferior cerebral vein level before the MCA bifurcated into frontaland parietal branches. The cranial skin incision is then closed. Ratpups are then placed in an incubator maintained at 37° C. until theyawoke, and are then transferred to their mother.

6 hours later inventive transporter cargo conjugate molecules comprisingTAT derived transporter constructs according to any of SEQ ID NOs: 8 to136 and JNK1 or IB1 derived cargo peptides according to any of SEQ IDNOs: 137 to 220 are injected intraperitoneally. 24 hours after thecoagulation, the rats are anesthetized with chloral hydrate and perfusedthrough the ascending aorta with 4% paraformaldehyde in PBS. Brains arethen removed and kept for 2 hours in the same fixative solution, andplaced in a gradient of 30% sucrose in PBS for about 15 hours at 4° C.Brains are frozen in isopentane (−40° C.) and stored at −20° C. Coronalcryostat sections of 50 □m are collected on glass slides. The sectionsare stained with cresyl violet. Each tenth section is analyzed and thetotal volume of the lesion is calculated using the Neuroleucidaprogramme.

19. Evaluation of Neuroprotection by Inventive Chimeric Peptides afteriv Administration Against Focal Cerebral Ischaemia, in a Transient MCAOModel

Transient ischemia in adult mice. Using male ICR-CD1 mice (6 weeks old;18-37 g; Harlan), ischemia is provoked by introducing a filament fromthe common carotid artery into the internal carotid and advancing itinto the arterial circle, thereby occluding the middle cerebral artery.The regional cerebral blood flow is measured by laser Doppler flowmetry,with a probe fixed on the skull throughout the ischemia until 10 minafter reperfusion. Rectal temperature is measured and maintained at 37°C. The mice are killed 48 h after reperfusion. Serial cryostat sections20 lam thick are traced using a computer-microscope system equipped withthe Neurolucida program (MicroBrightField) and the volumes of theischemic area and of the whole brain are calculated (blinded) with theNeuroexplorer program. The infarct volume sizes (mm³) after bolus ivadministration of placebo and inventive transporter cargo conjugatemolecules comprising TAT derived transporter constructs according to anyof SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargo peptides accordingto any of SEQ ID NOs: 137 to 220, 1.3 mg/kg 6 hours after reperfusion(30 minutes clamp) in an adult mice model is determined.

20. Assay on Neuronal Cultures by Measuring LDH Release Following NMDAStimulation

The neuroprotective effect of the inventive transporter cargo conjugatemolecules comprising TAT derived transporter constructs according to anyof SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargo peptides accordingto any of SEQ ID NOs: 137 to 220 is evaluated in sister culturespre-treated for 30 min with the indicated concentrations of peptides orMK-801 before continuous exposure to 100 μM NMDA. After 12 h of NMDAtreatment, in cultures pretreated with 5 μM of inventive transportercargo conjugate molecules comprising TAT derived transporter constructsaccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargopeptides according to any of SEQ ID NOs: 137 to 220 the degenerativechanges due to NMDA exposure, the morphological appearance, number anddistribution of the neurons are determined.

Cortical neuronal culture. Small pieces of cortex are dissected from thebrains of two day old rat pups, incubated with 200 units of papain for30 min at 34° C., and then the neurons are plated at densities ofapproximately 1×10⁶ cells/plate on dishes pre-coated with 100 μg/mlpoly-D-lysine. The plating medium consisted of B27/Neurobasal (LifeTechnologies, Gaithersburg, Md.) supplemented with 0.5 mM glutamine, 100U/ml penicillin and 100 ug/ml streptomycin.

Lactate dehydrogenate (LDH) cytotoxicity assay. LDH released into thebathing medium 12, 24 and 48 h after NMDA administration is measuredusing the Cytotox 96 non-radioactive cytotoxicity assay kit (Promega,Wis.).

21. Inhibition of Endogenous JNK Activity in HepG2 Cells Using an all-inOne Well Approach.

HepG2 cells are seeded at 3,000 cells/well the day prior the experiment.Then, increasing concentrations of either interleukin-1□ [IL-1β] ortumor necrosis factor α [TNFα] (a) are added to activate JNK for 30minutes. Cells are lysed in 20 mM Hepes, 0.5% Tween pH 7.4 and processedfor AlphaScreen JNK. (b) Z′ for the JNK activity induced by 10 ng/mlIL-1□ and measured in 384 wells/plate (n=96). (c) Inhibition ofendogenous IL-1 β-induced JNK activity with chemical JNK inhibitors[staurosporin and SP600125]. (d) Effect of inventive inventivetransporter cargo conjugate molecules comprising a TAT derivedtransporter constructs according to SEQ ID NOs: 8 to 136 and peptidicinhibitors L-TAT-IB1(s) on IL-1α dependent JNK activity.

Methods: Alphascreen Kinase Assay

Principle: AlphaScreen is a non-radioactive bead-based technology usedto study biomolecular interactions in a microplate format. The acronymALPHA stands for Amplified Luminescence Proximity Homogenous Assay. Itinvolves a biological interaction that brings a “donor” and an“acceptor” beads in close proximity, then a cascade of chemicalreactions acts to produce an amplified signal. Upon laser excitation at680 nm, a photosensitizer (phthalocyanine) in the “donor” bead convertsambient oxygen to an excited singlet state. Within its 4 μsec half-life,the singlet oxygen molecule can diffuse up to approximately 200 nm insolution and if an acceptor bead is within that proximity, the singletoxygen reacts with a thioxene derivative in the “acceptor” bead,generating chemiluminescence at 370 nm that further activatesfluorophores contained in the same “acceptor” bead. The excitedfluorophores subsequently emit light at 520-620 nm. In the absence of anacceptor bead, singlet oxygen falls to ground state and no signal isproduced.

Kinase reagents (B-GST-cJun, anti P-cJun antibody and active JNK3) arefirst diluted in kinase buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl₂, 1 mMDTT, 100 μM Na₃VO₄, 0.01% Tween-20) and added to wells (15 μl).Reactions are then incubated in presence of 10 μM of ATP for 1 h at 23°C. Detection is performed by an addition of 10 μl of beads mix (ProteinA acceptor 20 μg/ml and Streptavidin donor 20 μg/ml), diluted indetection buffer (20 mM Tris-HCl pH 7.4, 20 mM NaCl, 80 mM EDTA, 0.3%BSA), followed by an another one-hour incubation at 23° C. in the dark.For measurement of JNK endogenous activity, kinase assays are performedas described above except active JNK3 is replaced by cells lysates andreaction kinase components are added after the cells lysis. B-GST-cjunand P-cJun antibody are used at the same concentrations whereas ATP isused at 50 μM instead of 10 μM. AlphaScreen signal is analyzed directlyon the Fusion or En Vision apparatus.

22. Treatment of Noise Trauma

Inventive transporter cargo conjugate molecules comprising TAT derivedtransporter constructs according to any of SEQ ID NOs: 8 to 136 and JNK1or IB1 derived cargo peptides according to any of SEQ ID NOs: 137 to 220are applied onto the round window membrane of the cochlea of 3 groups ofguinea pigs (each group with 6 animals) in 2 microliters of a gelformulation of 2.6% buffered hyaluronic acid (Hylumed, Genzyme Corp.) ata concentration of 100 □M either 30 minutes before noise trauma (120 dBat 6 kHz during 30 minutes) or 30 minutes or 4 hours thereafter.Untreated ears served as control. Hearing threshold shifts are evaluatedby auditory brainstem response measurements 20 minutes after noisetrauma (temporary threshold shift, TTS) and 15 days following the trauma(permanent threshold shift, PTS). Administration of D-TAT-IB1(s)protected against permanent hearing loss even if applied after exposureto excessive noise compared to non-treated ears.

23. Evaluation of the Therapeutical Activity of Inventive TransporterCargo Conjugate Molecules Comprising TAT Derived Transporter ConstructsAccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 Derived CargoPeptides According to any of SEQ ID NOs: 137 to 220 in the Treatment ofColitis

a) Test System:

-   -   i) Species/Strain: Mouse/BALB/c    -   ii) Source: Harlan Israel, Ltd.    -   i) Gender: Female    -   iv) Total No. of Animals: n=150    -   v) Age: Young adults, 7 weeks of age at study initiation    -   vi) Body Weight: Weight variation of animals at the time of        treatment initiation does not exceed ±20% of the mean weight.    -   vii) Animals Health: The health status of the animals used in        this study is examined on arrival, only animals in good health        are acclimatized to laboratory conditions (at least seven days)        and are used in the study.    -   viii) Randomization: Animals are randomly assigned to        experimental groups according to a Table of Random Numbers.    -   ix) Termination: At the end of the study surviving animals are        euthanized by cercical dislocation.

b) Test Procedures

-   -   Colitis is induced by administration of TNBS dissolved in 50%        Ethanol    -   All animals are then treated with doses of inventive transporter        cargo conjugate molecules comprising TAT derived transporter        constructs according to any of SEQ ID NOs: 8 to 136 and JNK1 or        TM derived cargo peptides according to any of SEQ ID NOs: 137 to        220 in the range of 0.1 to 1000 μg/kg, either intraperitoneally        or subcutaneously, as a single or repeated daily doses (see        above).

c) Observations and Examinations

i) Clinical Signs

Throughout the duration of the above experiment, careful clinicalexaminations are carried out and recorded. Observations included changesexternal appearance, e.g. of the skin, fur, eyes, mucous membranes,occurrence of secretions and excretions (e.g. diarrhea), and autonomicactivity. Changes in gait, posture and response to handling, as well asthe presence of bizarre behavior, tremors, convulsions, sleep and comaare also noted.

ii) Body Weights

Determination of individual body weight of animals is made on a dailybasis.

iii) Clinical Assessment of Colitis

Body weight, stool consistency and bleeding per rectum are all recordeddaily and served as the parameters of disease severity score:

Weight Stool Presence of blood Score loss (%) consistency per rectum 0None Normal Negative 1 1-5 Redness, swelling Negative of the anus 2 5-10 Loose stool Negative 3 10-15 Diarrhea Negative 4 >15 DiarrheaBleeding 5 Death

iv) Gross Pathology of the Colon

On the last day of the experiment, animals are euthanized and the colonis removed for gross pathology evaluation according to the followingscore:

Grade Signs 0 No abnormalities detected 1 Edema and redness on onelocation 2 Edema and redness on more than one location, or a verymassive endema and redness capture more than 50% of the colon 3 Oneulcer 4 More than one ulcer or a very long severe ulcer24. Determining the Activity of Inventive Transporter Cargo ConjugateMolecules Comprising TAT Derived Transporter Constructs According to anyof SEQ ID NOs: 8 to 136 and JNK1 or IB1 Derived Cargo Peptides Accordingto any of SEQ ID NOs: 137 to 220 in the Treatment of Viral InfectionsVaricella-Zoster Virus (VZV)

Determination of the activity of inventive transporter cargo conjugatemolecules comprising TAT derived transporter constructs according to anyof SEQ ID NOs: 8 to 136 and JNK1 or IB I derived cargo peptidesaccording to any of SEQ ID NOs: 137 to 220 in cultured host cells (humanforeskin fibroblasts (HFFs)). Viruses are obligate intracellularparasites that require a functional cell environment to complete theirlifecycle; dying cells do not support virus replication. Additionally,inhibitors of cell functions may be toxic to cells, which couldnon-specifically prevent virus growth. Thus, sick or dying host cellscould exhibit nonspecifically reduced virus titers. Since this mayfalsify the results, a cytotoxicity assay is carried out first,determining the tolerance of the cultured cells to the test compound.Subsequently, a plaque reduction assay is carried out and then activityof the inventive transporter cargo conjugate molecules comprising TATderived transporter constructs according to any of SEQ ID NOs: 8 to 136and JNK1 or IB I derived cargo peptides according to any of SEQ ID NOs:137 to 220 is tested with respect to Viral Zoster Virus (VZV) ininfected cells.

A) Determination of the Cytotoxicity of Inventive Transporter CargoConjugate Molecules Comprising TAT Derived Transporter ConstructsAccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 Derived CargoPeptides According to any of SEQ ID NOs: 137 to 220:

For determination of toxicity, cultured cells (human foreskinfibroblasts (HFFs)) are seeded in 96-well tissue culture plates. Mediumcontaining DMSO (same level as 5 □M inventive transporter cargoconjugate molecules comprising TAT derived transporter constructsaccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargopeptides according to any of SEQ ID NOs: 137 to 220) is added at severalconcentrations of (1, 2, and 5 □M) for 24 h. Subsequently, a Neutral Redassay is carried out. Neutral Red colorimetric assays for cytotoxicityassays (in sets of 6 replicates) are used to set the maximum dose forsubsequent efficacy assays (as performed in Taylor et al, 2004, J.Virology, 78:2853-2862). Live cells absorb Neutral Red and, accordingly,the level of absorbance is a quantitative measure of cell viability andnumber. Neutral Red uptake is directly proportional to the number ofcells and also reflects normal endocytosis. Therefore, a brief pulse ofNeutral Red is added to the medium at 0 or 24 hours. After fixation andextraction, dye is added and the amount of dye in each sample ismeasured in an ELISA plate reader at 540 nm.

B) Plaque Reduction Assay to Evaluate the Antiviral Effects of InventiveTransporter Cargo Conjugate Molecules Comprising TAT Derived TransporterConstructs According to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1Derived Cargo Peptides According to any of SEC) ID NOs: 137 to 220Against Varicella-Zoster Virus (VZV):

To determine whether inventive transporter cargo conjugate moleculescomprising TAT derived transporter constructs according to any of SEQ IDNOs: 8 to 136 and JNK1 or IB1 derived cargo peptides according to any ofSEQ ID NOs: 137 to 220 exhibit a dose-dependent antiviral effect, arange of concentrations surrounding the standard 1 □M dose are tested.In this plaque reduction assay, confluent human foreskin fibroblasts(HFFs) in 24-well plates are inoculated with VZV-infected HFFs at aratio of 1:100 (multiplicity of infection MOI=0.01) and adsorbed to thecells for 2 hours. The excess virus is ished out, and medium containing0 (DMSO only), 0.5, 1, or 2 inventive transporter cargo conjugatemolecules comprising TAT derived transporter constructs according to anyof SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargo peptides accordingto any of SEQ ID NOs: 137 to 220 is added. One sample is taken at thistime to measure the initial level of infection; wherein each wellcontained ˜150 pfu. After 24 hours, duplicate wells are trypsinized, andthen the cell suspensions are titered on MeWo cell monolayers intriplicate to determine the number of VZV-infected cells in each sample.During unrestricted growth, VZV usually increases by 10-fold over 1 daybecause it propagates by cell-cell spread. This is what is observed forthe cultures treated with DMSO alone, which yielded 1200±430 pfu. Thecytotoxicity and efficacy data are determined. From these data, apreliminary Selective Index (Tox/EC₅₀) of 5.0 □M/0.3 □M is calculated.

C) Measurement of Varicella-Zoster Virus (VZV) Replication in HumanForeskin Fibroblasts (HFFs) with Inventive Transporter Cargo ConjugateMolecules Comprising TAT Derived Transporter Constructs According to anyof SEQ ID NOs: 8 to 136 and JNK1 or IB1 Derived Cargo Peptides Accordingto any of SEQ ID NOs: 137 to 220:

To determine the minimum effective dose of inventive transporter cargoconjugate molecules comprising TAT derived transporter constructsaccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargopeptides according to any of SEQ ID NOs: 137 to 220 that preventsvaricella-zoster virus (VZV) replication inhuman foreskin fibroblasts(HFFs) confluent monolayers of HFFs are inoculated with VZV-BAC-Lucstrain for 2h, then treated for 24 h with inventive transporter cargoconjugate molecules comprising TAT derived transporter constructsaccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargopeptides according to any of SEQ ID NOs: 137 to 220 inconcentrations of0.25, 0.5, or 1.0 □M or with the a negative control (DAK, 1.0 □M). Virusyield is measured by luciferase assay. Samples are in triplicate and theaverage luminescence is shown; error bars represent the standarddeviation of the mean.

25. Determining the Activity of Inventive Transporter Cargo ConjugateMolecules Comprising TAT Derived Transporter Constructs According to anyof SEQ ID NOs: 8 to 136 and JNK1 or IB1 Derived Cargo Peptides Accordingto any of SEQ ID NOs: 137 to 220 in the Treatment of Chronic ObstructivePulmonary Disease (COPD)

In order to determine the activity of the exemplary inventivetransporter cargo conjugate molecules comprising TAT derived transporterconstructs according to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1derived cargo peptides according to any of SEQ ID NOs: 137 to 220 in thetreatment of Chronic Obstructive Pulmonary Disease (COPD) theseinventive transporter cargo conjugate molecules are used in an animalmodel of Bleomycin induced acute lung inflammation and fibrosis. Theprotocol of bleomycin induced inflammation and fibrosis has beendescribed before in the literature. The aim of the Experiment is toinvestigate the effect of these inventive transporter cargo conjugatemolecules by subcutaneous (s.c.) route on neutrophil recruitment inbroncho alveolar lavage (BAL) and lung in bleomycin induced inflammationand fibrosis:

-   -   at 1 day after a single bleomycin administration (10 mg/kg)    -   and at day 10 with the development of fibrosis

1) Method and Experimental Approach

The test compounds selected from inventive transporter cargo conjugatemolecules comprising TAT derived transporter constructs according to anyof SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargo peptides accordingto any of SEQ ID NOs: 137 to 220 at two doses and vehicle control aregiven s.c. with a single intranasal administration of bleomycin and miceare analyzed after 1 and 10 days. The animals used in the model are 10C57BL/6 mice (8 weeks old) per group. The experimental groups includevehicle, 0.001 mg/kg of inventive transporter cargo conjugate moleculescomprising TAT derived transporter constructs according to any of SEQ IDNOs: 8 to 136 and JNK1 or IB1 derived cargo peptides according to any ofSEQ ID NOs: 137 to 220 and 0.1 mg/kg of these inventive transportercargo conjugate molecules, and the treatment consists of repeatedsub-cutaneous administration of these inventive transporter cargoconjugate molecules prior to bleomycin administration every 3 days.Acute lung inflammation at 24h is monitored by BAL lavage, cytology,cell counts, and lung myeloperoxidase activity. The effect of thecompound is compared with vehicle controls. Lung fibrosis is assessedhistologically using hematoxylin and eosin staining at day 10 after thesingle dose of bleomycin.

1.1) Bleomycin Administration

Bleomycin sulfate in saline (10 mg/kg body weight) from BellonLaboratories (Montrouge, France) or saline are given through the airwaysby nasal instillation in a volume of 40 μL under light ketamine-xylasineanesthesia. The groups for Bleomycin administration for both bleomycininduced inflammation and fibrosis included: Vehicle, 0.001 mg/kg of theinventive transporter cargo conjugate molecules comprising TAT derivedtransporter constructs according to any of SEQ ID NOs: 8 to 136 and JNK1or IB1 derived cargo peptides according to any of SEQ ID NOs: 137 to 220and 0.1 mg/kg of these inventive transporter cargo conjugate molecules.The route for bleomycin induced inflammation is subcutaneous (s.c.)route, and administration occurrs as a single dose. The route forbleomycin induced fibrosis is subcutaneous (s.c.) route, andadministration occurred 3 times in 10 days.

1.2) Bronchoalveolar Lavage Fluid (BALF)

After incision of the trachea, a plastic cannula is inserted andairspaces are ished using 0.3 ml of PBS solution, heated to 37° C. Thesamples collected are dispatched in 2 fractions: the first one (1 mlcorresponding to the 2 first lavages) is used for mediator measurementand the second one for the cell determination (4 ml). The first fractionis centrifuged (600 g for 10 min) and supernatant is fractionated andkept at −80° C. until mediator determination. The cell pellet is thenresuspended in 0.4 ml sterile NaCl, 0.9%, and pooled with the secondfraction and is used for cell counts.

1.3) Lung Homogenization

After BAL the whole lung is removed and placed inside a microtube(Lysing matrix D, Q Bio Gene, Illkrich, France) with 1 ml of PBS, totallung tissue extract is prepared using a Fastprep® system (FP 120, Q BioGene, Illkrich, France), the extract is then centrifuged and thesupernatant stored at −80° C. before mediator measurement and collagenassay with Sircol Collagen Assay (France Biochem Division, France).

1.4) Cell Count and Determination

Total cell count is determined in BAL fluid using a Malassezhemocytometer. Differential cell counts are performed on cytospinpreparations (Cytospin 3, Thermo Shandon) after staining with MGGDiff-quick (Dade Behring AG). Differential cell counts are made on 200cells using standard morphological criteria.

1.5) TNF Measurement

TNF level in BALF is determined using ELISA assay kits (Mouse DuoSet,R&D system, Minneapolis, USA) according to manufacturer's instructions.Results are reported as pg/ml.

1.6) MPO-Measurement

MPO-levels are measured upon administration of inventive transportercargo conjugate molecules comprising TAT derived transporter constructsaccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargopeptides according to any of SEQ ID NOs: 137 to 220.

1.7) Histology

After BAL and lung perfusion, the large lobe is fixed in 4% bufferedformaldehyde for standard microscopic analysis. 3 μm sections arestained with hematoxylin and eosin (H&E).

26. Determining the Activity of Inventive Transporter Cargo ConjugateMolecules Comprising TAT Derived Transporter Constructs According to anyof SEQ ID NOs: 8 to 136 and JNK1 or IB1 Derived Cargo Peptides Accordingto any of SEQ ID NOs: 137 to 220 in the Treatment of Alzheimer's Disease

In order to determine the activity of the exemplary inventivetransporter cargo conjugate molecules comprising TAT derived transporterconstructs according to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1derived cargo peptides according to any of SEQ ID NOs: 137 to 220 inAlzheimer's disease, these peptides are evaluated in the hAPP-transgenicmice model overexpressing APP751 with London and Swedish mutations usingthe behavioral Morris Water Maze test as well as immunohistologicaltests measuring plaque load and ELISA tests measuring ß-amyloid₁₋₄₀ andß-amyloid₁₋₄₂ levels in the brain of mice.

a) Methods

i) Introduction

The study is designed to evaluate the efficacy of the inventivetransporter cargo conjugate molecules comprising TAT derived transporterconstructs according to any of SEQ ID NOs: 8 to 136 and JNK1 or IBderived cargo peptides according to any of SEQ ID NOs: 137 to 220 onbehavioral, biochemical and histological markers using 5 months (±2weeks) old female hAPP Tg mice. Therefore, mice are treated every two orthree weeks up to 4 months and in the end of the treatment periodbehavior is evaluated in the Morris Water Maze. At sacrifice brain, CSFand blood are collected. Aß40 and Aß42 levels are determined in fourdifferent brain homogenate fractions as well as in CSF of Tg mice.Plaque load is quantified in the cortex and the hippocampus of 8 Tganimals per treatment group.

ii) Animals

Female Tg mice with a C57BL/6×DBA background and an age of 5 months (±2week) are randomly assigned to treatment groups 1 to 3 (n=12). Animalsare subjected to administration of vehicle or inventive transportercargo conjugate molecules comprising TAT derived transporter constructsaccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargopeptides according to any of SEQ ID NOs: 137 to 220 in two differentconcentrations beginning at 5 months of age and continued for up to 4months with subcutaneous (s.c.) applications every second or third week.All animals which are used for the present study had dark eyes and arelikely to perceive the landmarks outside the MWM pool. However, it hadto be excluded that seeing abilities of an animal are poor, which iscontrolled in the visible platform training, the so called pretest,before treatment start for all animals including reserves enclosed tothe study. In case a seeing handicap for a specific animal would havebeen affirmed, the mouse would have been excluded from the study.

iii) Animal Identification and Housing

Mice are individually identified by ear markings. They are housed inindividual ventilated cages (IVCs) on standardized rodent beddingsupplied by Rettenmaier®. Each cage contained a maximum of five mice.Mice are kept according to the JSW Standard Operating Procedures (SOPGEN011) written on the basis of international standards. Each cage isidentified by a colored card indicating the study number, sex, theindividual registration numbers (IRN) of the animals, date of birth, aswell as the screening date and the treatment group allocation. Thetemperature during the study is maintained at approximately 24° C. andthe relative humidity is maintained at approximately 40-70%. Animals arehoused under a constant light-cycle (12 hours light/dark). Normal tapwater is available to the animals ad libitum.

iv) Treatment

Forty female hAPP transgenic mice are treated with either 0.1 mg/kgb.w./every two weeks or 10 mg/kg b.w./every three weeks of inventivetransporter cargo conjugate molecules comprising TAT derived transporterconstructs according to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1derived cargo peptides according to any of SEQ ID NOs: 137 to 220 in twodifferent dosages (n=12/group) or treated with the vehicle (n=12) s.c.once every three weeks over four months.

v) Morris Water Maze (MWM)

The Morris Water Maze (MWM) task is conducted in a black circular poolof a diameter of 100 cm. Tap water is filled in with a temperature of22±1° C. and the pool is virtually divided into four sectors. Atransparent platform (8 cm diameter) is placed about 0.5 cm beneath thewater surface. During the whole test session, except the pretest, theplatform is located in the southwest quadrant of the pool. One daybefore the 4 days lasting training session animals had to perform a socalled “pre-test” (two 60 sec lasting trials) to ensure that the seeingabilities of each animal are normal. Only animals that fulfilled thistask are enclosed to the MWM testing. In the MWM task each mouse had toperform three trials on four consecutive days. A single trial lasted fora maximum of maximum one minute. During this time, the mouse had thechance to find the hidden, diaphanous target. If the animal could notfind a “way” out of the water, the investigator guided to or placed themouse on the platform. After each trial mice are allowed to rest on theplatform for 10-15 sec. During this time, the mice had the possibilityto orientate in the surrounding. Investigations took place under dimmedlight conditions, to prevent the tracking system from negativeinfluences (Kaminski; PCS, Biomedical Research Systems). On the wallssurrounding the pool, posters with black, bold geometric symbols (e.g. acircle and a square) are fixed which the mice could use the symbols aslandmarks for their orientation. One swimming group per trial consistsof five to six mice, so that an intertrial time of about five to tenminutes is ensured. For the quantification of escape latency (the time[second]—the mouse needs to find the hidden platform and therefore toescape from the water), of pathway (the length of the trajectory [meter]to reach the target) and of the abidance in the goal quadrant acomputerized tracking system is used. The computer is connected to acamera placed above the centre of the pool. The camera detected thesignal of the light emitting diode (LED), which is fixed with a littlehairgrip on the mouse's tail. One hour after the last trial on day 4 themice had to fulfill a so-called probe trial. At this time, the platformis removed from the pool and during the one-minute probe trial; theexperimentator counts the number of crossings over the former targetposition. Additionally the abidance in this quadrant as well as thethree other quadrants is calculated. Through out this trial a mousecould not get any, howsoever-natured, clue from the platform.

vi) Tissue Sampling

At the end of the treatment period, and following all behavioraltesting, all remaining mice (n=28) are sacrificed. Therefore, all miceare sedated by standard inhalation anesthesia (Isofluran, Baxter) asdescribed in SOP MET030. Cerebrospinal fluid (CSF) is obtained by bluntdissection and exposure of the foramen magnum. Upon exposure, a Pasteurpipette is inserted to the approximate depth of 0.3-1 mm into theforamen magnum. CSF is collected by suctioning and capillary actionuntil flow fully ceases. Two aliquots of each sample are immediatelyfrozen and kept at −80° C. until ready for further analysis with ELISAtechnique. After CSF sampling, each mouse is placed in dorsalrecumbence, thorax is opened and a 26-gauge needle attached to a 1 ccsyringe is inserted into the right cardiac ventricular chamber. Lightsuction is applied to the needle and blood is collected into EDTA andconsequently used to obtain plasma. To get plasma, blood samples fromeach mouse are spun at 1,750 rpm (700 g) for 10 minutes in a centrifuge(GS—6R Beckman) using a rotor with swing buckets (GH—3.8 Beckman).Plasma is frozen and stored at −20° C. until further analysis. Afterblood sampling transgenic mice are intracardially perfused with 0.9%sodium chloride. Brains are rapidly removed the cerebellum is cut off.The right hemispheres of all mice are immersion fixed in freshlyproduced 4% Paraformaldehyde/PBS (pH 7.4) for one hour at roomtemperature. Thereafter brains are transferred to a 15% sucrose PBSsolution for 24 hours to ensure cryoprotection. On the next day brainsare frozen in isopentane and stored at −80° C. until used forhistological investigations (SOP MET042). The left hemispheres areweighed and frozen in liquid nitrogen and stored at −80° C. forbiochemical analysis.

vii) Determination of Aß₁₋₄₀ and Aß₁₋₄₂

In four different brain homogenate fractions of each Tg mouse as well asin CSF samples the Aß₁₋₄₀ and Aß₁₋₄₂ levels are evaluated with ELISAtechnique. Highly sensitive Aß₁₋₄₀ and Aß₁₋₄₂ ELISA test kits arepurchased from The Genetics Company™, Switzerland (SOP MET058). CSF isprepared as described above. For the brain homogenates frozenhemispheres are homogenized in TRIS buffered saline (TBS)—buffer (5 ml)containing protease inhibitor cocktail. 1.25 ml of this initial brainTBS homogenate is stored at −80° C., 1.25 ml have been furtherinvestigatated. The remaining brain homogenate (2.5 ml) is centrifugedand the resulting supernatant (=TBS fraction) is aliquoted and kept at−20° C. until ELISA determination. The pellet is suspended in TritonX-100 (2.5 ml), centrifuged and the supernatant (=Triton X-100 fraction)is aliquoted and kept at −20° C. These steps are repeated with SDS (2.5ml). The pellet out of the SDS fraction is suspended in 70% formic acid(0.5 ml) prior to subsequent centrifugation. The obtained supernatant isneutralized with 1 M TRIS (9.5 ml) aliquoted and kept at −20° C. (=FAfraction). Samples of the four brain homogenate fraction (TBS, TritonX-100, SDS, and FA) are used for Aß₁₋₄₀ and Aß₁₋₄₂ determination withELISA technique. ELISA test kits are purchased from The GeneticsCompany™, Switzerland (SOP MET062). It could be assumed that TBS andTriton X-1130 solubilize monomeric to oligomeric structures. Polymerslike protofibrils and water insoluble fibrils could be dissolved in SDSand FA. In this regard the investigation of all four fractions alsoprovides insight in A□ polymerization status.

viii) Evaluation of Brain Morphology

Brain tissues of all Tg animals investigated are handled in exactly thesame way to avoid bias due to variation of this procedure. From brainhalves of 24 Tg mice (8 of each group) 20 cryo-sections per layer(altogether 5 layers), each 10 □m thick (Leica CM 3050S) are sagittallycut and 5 (one from each layer) are processed and evaluated forquantification of plaque load. The five sagittal layers correspondedwith the FIGS. 104 to 105, 107 to 108, 111 to 112, 115 to 116 and 118 to119 according to the morphology atlas “The Mouse Brain” from Paxinos andFranklin (2nd edition). The first layer is specified by the requirementto include the whole hippocampus with it's regions CA1, CA2, CA3, GDlband GDmb. Immunoreactivity is quantitatively evaluated in thehippocampus and in the cortex using the monoclonal human Aß-specificantibody 6E10 (Signet) as well as ThioflavinS staining. Remaining brainhemispheres or tissue not used are saved and stored at JSW CNS until theend of the project.

b) Evaluation

i) Behavior

In the Morris Water Maze trials length of swimming path, escapelatencies, swimming speed and in the probe trial crossings over theformer platform position and the time spent in each quadrant of the poolare measured for each Tg animal with a special computer software.

ii) Biochemical Evaluation

From all Tg mice CSF samples as well as samples from the brainpreparations are analyzed with commercially available Aß₁₋₄₀ and Aß₁₋₄₂ELISAs. Measurements of adequate standards are performed concurrently.Samples from brain preparations are analyzed in duplicates. Due to thesmall sample amount CSF samples are analyzed in a single measurementonly.

iii) Histology

ii) Measurement of Amyloid Depositions and Plaque Load

For 6E10 immunohistochemistry the following evaluation procedure isused:

-   -   aa) Contrasting the image for visualization of slice borders        without applying the contrast on the image.    -   bb) Interactive drawing of the cortical outlines and the        following measurement of the cortical area (=region area).    -   cc) Interactive drawing of the area of interest (AOI), in which        stained objects are detected over a certain intensity based        threshold level (the same for each image) and above a size of 8        □m².    -   dd) Measurement of the area of each object, the sum of stained        area in the AOI as well as the number of objects after a smooth        contrasting to enhance signal/noise ratio (the same for each        image).    -   ee) Repetition of aa)-dd) for the hippocampus.    -   ff) Calculation of the mean plaque size (=“sum area of        plaques/number of plaques”), the relative plaque number and area        (=“number of plaques/region area” and “sum area of        plaques/region area*100”).    -   gg) Automated data export into an Excel spread sheet, including        the parameters “image title, region area, number of plaques, sum        of plaque area, relative plaque number, relative plaque area and        mean plaque size. A field for remarks is used to record image        quality and exclusion criteria, respectively. Exclusion criteria        are missing parts of the slice, many wrinkles, dominant flaws or        staining inconsistencies (e.g. due to bulges, which can impede        the full reaction of the blocking reagent).    -   hh) Closing the image without saving (to keep raw data raw).        27. Determining the Activity of Inventive Transporter Cargo        Conjugate Molecules Comprising TAT Derived Transporter        Constructs According to any of SEQ ID NOs: 8 to 136 and JNK1 or        IB1 Derived Cargo Peptides According to any of SEQ ID NOs: 137        to 220 in the Treatment of Diabetes Type 2

This is designed to determine the activity of inventive transportercargo conjugate molecules comprising TAT derived transporter constructsaccording to any of SEQ ID NOs: 8 to 136 and JNK1 or IB1 derived cargopeptides according to any of SEQ ID NOs: 137 to 220 in the treatment ofDiabetes Type 2, particularly to determine the effect of chronictreatment with these inventive transporter cargo conjugate molecules inthe db/db mice model of type 2 diabetes by evaluating fasting bloodglucose levels every third day (28 days)

a) Materials and Methods

i) Animals

A total of twenty (20) male db/db mice (8 weeks old) are obtained fromCharles River (Germany). Upon arrival, animals are group housed(n=6-7/group) and offered regular rodent chow (Altromin standard #1324chow; C. Petersen, Ringsted, Denmark) and water ad libitum unlessotherwise stated.

The mice are housed under a 12:12 L/D cycle (lights on at 4:00 andlights off at 16:00) and in temperature and humidity controlled rooms.

ii) Groups and Randomization

On day −4, mice are randomized according to blood glucose level (fasted;blood glucose measured on Biosen S line analyzer (EKF diagnostic,Germany) to participate in one of the following drug treatment groups(n=6):

-   -   1) Vehicle control, S.C. (physiological saline)    -   2) inventive transporter cargo conjugate molecules comprising        TAT derived transporter constructs according to any of SEQ ID        NOs: 8 to 136 and JNK1 or IB1 derived cargo peptides according        to any of SEQ ID NOs: 137 to 220; 1 mg/kg; s.c.    -   3) inventive transporter cargo conjugate molecules comprising        TAT derived transporter constructs according to any of SEQ ID        NOs: 8 to 136 and JNK1 or IB1 derived cargo peptides according        to any of SEQ ID NOs: 137 to 220; 10 mg/kg; s.c

All doses listed are calculated for the free-base. Drug purity: 95.28%,peptide content: 78.0%. All compounds are administered sub-cutaneously(s.c.) in a volume of 3 ml/kg. The formulation instructions for vehiclecontrol and inventive transporter cargo conjugate molecules comprisingTAT derived transporter constructs according to any of SEQ ID NOs: 8 to136 and JNK1 or IB1 derived cargo peptides according to any of SEQ IDNOs: 137 to 220 are as follows:

First, inventive transporter cargo conjugate molecules comprising TATderived transporter constructs according to any of SEQ ID NOs: 8 to 136and JNK1 or IB1 derived cargo peptides according to any of SEQ ID NOs:137 to 220 are dissolved in the vehicle. The formulations(concentrations of 0.33 and 3.3 mg/ml, corresponding to the doses of 1and 10 mg/kg, respectively) are prepared according to the proceduredetailed below. Concentrations are calculated and expressed taking intoaccount test items purity and peptide content (multiplier coefficient is1.346).

-   -   Preparation of a stock solution: the freeze-dried inventive        transporter cargo conjugate molecules comprising TAT derived        transporter constructs according to any of SEQ ID NOs: 8 to 136        and JNK1 or IB1 derived cargo peptides according to any of SEQ        ID NOs: 137 to 220 is thawed for one hour minimum and prepared        as a stock solution in the vehicle at 1 mM. Aliquots are        prepared for each treatment day and stored at approximately        −80° C. Dilutions of this stock solution to the required        concentrations are performed on each treatment day;    -   Storage of the stock solution: at approximately −80° C.;    -   Storage of the diluted preparations: at room temperature for 24        hours maximum.

Prior to solubilisation, the powder is stored at −20° C. The stabilityof the stock solution is 3 months at approximately −80° C.; thestability of the diluted formulations for animal dosing is 24 hours atroom temperature. Unused diluted material could be stored for up to 7days if kept at 4-8° C.

c) Experimental Procedure

Following 8 days of acclimatization the mice are treated daily at 08.00AM for 21 days by SC dosing 8 hours prior to lights out at 04.00 PMaccording to the outline groups.

i) Blood Glucose

Blood glucose is measured from 7 hour fasted animals 6 hours post dosingby collection of 10 μl blood samples from the tail-vein in hematocritetubes and subsequent analysis on a Biosen s-line analyzer(EKF-diagnostic; Germany).

ii) Metabolic Cages

Groups 1+3: Mice are placed in metabolic cages for the recording of24-hour food and water intake as well as 24-hour urine and faecesproduction. Mice are stratified into two sub-teams of n=6-7 andsubsequently the metabolic characterisation is performed.

iii) Adipokine Panel

Groups 1+3: On three occasions blood is collected from the tail veinusing EDTA coated hematocrite tubes (100 μl). Following centrifugationof blood the plasma is collected and stored at −20° C. untilmeasurement. Then, the following panel of adipokines/cytokines isdetermined using Luminex based 7-plex: leptin, resistin, MCP-1, PAI-1,TNF E, insulin and interleukin-6 (IL-6).

iv) Termination

Groups 1+3 (day 111): The following organs are excised and weighed:inguinal subcutaneous fat, epididymal fat, retroperitoneal fat, brain,liver, kidney, spleen and heart. All organs described above are samplesin 4% PFA for possible future histo-pathological examination. Also,pancreas (en bloc) is sampled for possible stereological andimunohistochemical analysis, and eyes are sampled for possible lateranalysis of retinopathy. Group 2 (day 28): No tissues or plasma arecollected.

28. TAT Derivatives Target Human Leukocyte Populations

Primary human white blood cells (WBC) were obtained from whole bloodafter red blood cell lysis. WBC were incubated with 1 uM of D-TAT (SEQID NO: 251)-FITC or r3-TAT (SEQ ID NO: 20)-FITC for 30 min at 37° C.,washed in acid buffer and stained with fluorescent antibodies againstcell type specific surface markers (CD₁₄ for monocytes, CD15 forpolymorphnuclears, CD3 for lymphocyte T, CD19 for lymphocyte B). Cellscontaining D-TAT-FITC and r3-L-TAT-FITC were finally analysed by flowcytometry to measure their respective transporter content. Both TATderivatives target the human leukocyte populations. dTAT and r3LTATbinds to monocytes, neutrophils and lymphocyte T cells, and lessefficiently to lymphocyte B cells. A minor difference between dTAT andr3-L-TAT specificity exists, D-TAT seeming to bind more efficiently tolymphocyte T than the r3-L-TAT.

29. Uptake of Selected Transporter Constructs According to the PresentInvention by Different Cell Types

Cells were plated in Poly-D-lysine pre-coated 96-well-plates atsubcontinent density (which can vary depending on the cell type used).Different FITC-coupled transporters were then incubated with the cells15 h at 3 μM. Following this time, cells were kept on ice for the restof the procedure. To remove cell-surface bound peptides, cells werefirst washed 2 Limes with an acid wash to remove plasma membrane-boundmolecules. Subsequently, cells were washed times with PBS and lysed in astandard lysis buffer for 30 min. Plates containing cell lysates werethen centrifuged for 5 min at 1500 rpm at 4° c. Clear supernatant wasthen collected and transferred into a black 96-well-plate for themeasure of intracellular FITC fluorescence. The following cells wereused:

-   Non leucocyte cell lines: HepG2: Flepatocarcinorna cells (human)    -   A549: Lung epithelial cells (human)-   Leucocyte cell lines: Raw: Macrophage cells (mouse)    -   177: Macrophage cells (mouse)-   Primary purified leucacytes: BMDM: Bone Marrow-Derived Macrophages    (mouse)

Results are expressed as percentage of D-TAT (SEQ ID NO: 251) (FIG. 17)or r3-L-TAT (SEQ ID NO: 20) (FIG. 18) uptake. All transporter constructsshow uptake in the respective cells, albeit at different rates.

1. A transport cargo conjugate molecule comprising a) a component (A)comprising a transporter construct comprising a sequence selected fromthe group consisting of an amino acid sequence comprising rKKRrQRRr (SEQID NO:20) and an amino acid sequence comprising rRRQrRKKr (SEQ IDNO:21), wherein lowercase designations indicate D-amino acids anduppercase designations indicate L-amino acids; and b) a component (B)comprising an effector molecule.
 2. The transporter cargo conjugatemolecule of claim 1, wherein the sequence is conjugated with atrafficking sequence selected from the group consisting of: a sequencederived from HIV-I TAT protein (SEQ ID NO: 8), a sequence derived froman amino acid sequence containing TAT residues 48-57 or 48 to 57 ofHIV-I TAT protein (SEQ ID NO: 8), a sequence derived from HSV VP22,obtained from Herpes simplex, a sequence derived from antennapediacarrier sequence, obtained from Drosophila antennapedia, FGF,lactoferrin, and a sequence derived from a basic peptide having a lengthof 5 to 15 amino acids, or 10 to 12 amino acids and comprising at least80%, or 85% or 90% basic amino acids selected from arginine, lysine andhistidine, or is selected from the group consisting of: arginine richpeptide sequences, HSV VP22, PTD-4 derived proteins or peptides,RGD-K₁₆, PEPT1, PEPT2, or proteins or peptides derived from PEPT1 orPEPT2, SynB3 or SynB3 derived proteins or peptides, protein convertase(PC) inhibitors, P21 derived proteins or peptides, and JNK1 derivedproteins or peptides.
 3. The transporter cargo conjugate molecule ofclaim 1, wherein the sequence is conjugated with one of the followingsequences or a reverse sequence thereof, SEQUENCE/ SEQ PEPTIDE NAME IDAA SEQUENCE TAT (1-86) 8 86 MEPVDPRLEP WKHPGSQPKT ACTNCYCKKC CFHCQVCFITKALGISYGRK KRRQRRRPPQ GSQTHQVSLS KQPTSQSRGD PTGPKE TAT (37-72) 9 36CFITKALGIS YGRKKRRQRR RPPQGSQTHQ VSLSKQ TAT (37-58) 10 22CFITKALGIS YGRKKRRQRR RP TAT (38-58)GGC 11 24 FITKALGISY GRKKRRQRRR PGGCTAT CGG(47-58) 12 15 CGGYGRKKRR QRRRP TAT (47-58)GGC 13 15YGRKKRRQRR RPGGC TAT (1-72) Mut 14 56 MEPVDPRLEP WKHPGSQPKT Cys/Ala 72AFITKALGIS YGRKKRRQRR RPPQGSQTHQ VSLSKQ L-TAT (sla) 17 10 GRKKRRQRRRL-TAT (slb) 18 9 RKKRRQRRR L-TAT (slc) 19 11 YDRKKRRQRRR r₃-L-TAT 20 9rKKRrQRRr r₃-L-TATi 21 9 rRRQrRKKr βA-r₃-L-TAT 22 10 βA-rKKRrQRRrβA-r₃-L-TATi 23 10 βA-rRRQrRKKr FITC-βA-r₃-L-TAT 24 10 FITC-βA-rKKRrQRRrFITC-βA-r₃-L-TATi 25 10 FITC-βA-rRRQrRKKr TAT(s2-1) 26 9 rAKRrQRRrTAT(s2-2) 27 9 rKARrQRRr TAT(s2-3) 28 9 rKKArQRRr TAT(s2-4) 29 9rKKRrARRr TAT(s2-5) 30 9 rKKRrQARr TAT(s2-6) 31 9 rKKRrQRAr TAT(s2-7) 329 rDKRrQRRr TAT(s2-8) 33 9 rKDRrQRRr TAT(s2-9) 34 9 rKKDrQRRr TAT(s2-10)35 9 rKKRrDRRr TAT(s2-11) 36 9 rKKRrQDRr TAT(s2-12) 37 9 rKKRrQRDrTAT(s2-13) 38 9 rEKRrQRRr TAT(s2-14) 39 9 rKERrQRRr TAT(s2-15) 40 9rKKErQRRr TAT(s2-16) 41 9 rKKRrERRr TAT(s2-17) 42 9 rKKRrQERr TAT(s2-18)43 9 rKKRrQREr TAT(s2-19) 44 9 rFKRrQRRr TAT(s2-20) 45 9 rKFRrQRRrTAT(s2-21) 46 9 rKKFrQRRr TAT(s2-22) 47 9 rKKRrFRRr TAT(s2-23) 48 9rKKRrQFRr TAT(s2-24) 49 9 rKKRrQRFr TAT(s2-25) 50 9 rRKRrQRRr TAT(s2-26)51 9 rKRRrQRRr TAT(s2-27) 52 9 rKKKrQRRr TAT(s2-28) 53 9 rKKRrRRRrTAT(s2-29) 54 9 rKKRrQKRr TAT(s2-30) 55 9 rKKRrQRKr TAT(s2-31) 56 9rHKRrQRRr TAT(s2-32) 57 9 rKHRrQRRr TAT(s2-33) 58 9 rKKHrQRRr TAT(s2-34)59 9 rKKRrHRRr TAT(s2-35) 60 9 rKKRrQHRr TAT(s2-36) 61 9 rKKRrQRHrTAT(s2-37) 62 9 rIKRrQRRr TAT(s2-38) 63 9 rKIRrQRRr TAT(s2-39) 64 9rKKIrQRRr TAT(s2-40) 65 9 rKKRrIRRr TAT(s2-41) 66 9 rKKRrQIRr TAT(s2-42)67 9 rKKRrQRIr TAT(s2-43) 68 9 rLKRrQRRr TAT(s2-44) 69 9 rKLRrQRRrTAT(s2-45) 70 9 rKKLrQRRr TAT(s2-46) 71 9 rKKRrLRRr TAT(s2-47) 72 9rKKRrQLRr TAT(s2-48) 73 9 rKKRrQRLr TAT(s2-49) 74 9 rMKRrQRRr TAT(s2-50)75 9 rKMRrQRRr TAT(s2-51) 76 9 rKKMrQRRr TAT(s2-52) 77 9 rKKRrMRRrTAT(s2-53) 78 9 rKKRrQMRr TAT(s2-54) 79 9 rKKRrQRMr TAT(s2-55) 80 9rNKRrQRRr TAT(s2-56) 81 9 rKNRrQRRr TAT(s2-57) 82 9 rKKNrQRRr TAT(s2-58)83 9 rKKRrNRRr TAT(s2-59) 84 9 rKKRrQNRr TAT(s2-60) 85 9 rKKRrQRNrTAT(s2-61) 86 9 rQKRrQRRr TAT(s2-62) 87 9 rKQRrQRRr TAT(s2-63) 88 9rKKQrQRRr TAT(s2-64) 89 9 rKKRrKRRr TAT(s2-65) 90 9 rKKRrQQRr TAT(s2-66)91 9 rKKRrQRQr TAT(s2-67) 92 9 rSKRrQRRr TAT(s2-68) 93 9 rKSRrQRRrTAT(s2-69) 94 9 rKKSrQRRr TAT(s2-70) 95 9 rKKRrSRRr TAT(s2-71) 96 9rKKRrQSRr TAT(s2-72) 97 9 rKKRrQRSr TAT(s2-73) 98 9 rTKRrQRRr TAT(s2-74)99 9 rKTRrQRRr TAT(s2-75) 100 9 rKKTrQRRr TAT(s2-76) 101 9 rKKRrTRRrTAT(s2-77) 102 9 rKKRrQTRr TAT(s2-78) 103 9 rKKRrQRTr TAT(s2-79) 104 9rVKRrQRRr TAT(s2-80) 105 9 rKVRrQRRr TAT(s2-81) 106 9 rKKVrQRRrTAT(s2-82) 107 9 rKKRrVRRr TAT(s2-83) 108 9 rKKRrQVRr TAT(s2-84) 109 9rKKRrQRVr TAT(s2-85) 110 9 rWKRrQRRr TAT(s2-86) 111 9 rKWRrQRRrTAT(s2-87) 112 9 rKKWrQRRr TAT(s2-88) 113 9 rKKRrWRRr TAT(s2-89) 114 9rKKRrQWRr TAT(s2-90) 115 9 rKKRrQRWr TAT(s2-91) 116 9 rYKRrQRRrTAT(s2-92) 117 9 rKYRrQRRr TAT(s2-93) 118 9 rKKYrQRRr TAT(s2-94) 119 9rKKRrYRRr TAT(s2-95) 120 9 rKKRrQYRr TAT(s2-96) 121 9 rKKRrQRYr r₃R₆ 1229 rRRRrRRRr L-R₉ 123 9 RRRRRRRRR L-R₈ 124 8 RRRRRRRR L-R₇ 125 7 RRRRRRRL-R₆ 126 6 RRRRRR L-R₅ 127 5 RRRRR PTD-4 128 11 YARAAARQARAPTD-4 (variant 1) 129 11 WARAAARQARA PTD-4 (variant 2) 130 11WARAQRAAARA L-P1 Penetratin 131 16 RQVKVWFQNRRMKWKK D-P1 Penetratin 13216 KKWKMRRNQFWVKVQR JNKI, bestfit 133 17 WKRAAARKARAMSLNLF JNKI, bestfit134 17 WKRAAARAARAMSLNLF (variant 1) MDCK transcytose 135 9 RYRGDLGRRsequence YKGL 136 4 YKGL

wherein lowercase designations indicate D-amino acids and uppercasedesignations indicate L-amino acids and FITC is fluoresceinisothiocyanate.
 4. The transporter cargo conjugate molecule of claim 2,wherein the arginine rich peptide sequence comprises 5, 6, 7, 8, or 9arginines.
 5. The transporter cargo conjugate molecule of claim 2,wherein the sequence is conjugated to a trafficking sequence derivedfrom HIV-1 TAT protein having a Tyrosine (Y) at position 2 of the TATderived sequence.
 6. The transporter cargo conjugate molecule of claim5, wherein the trafficking sequence derived from HIV-1 TAT proteinhaving a Tyrosine (Y) at position 2 of the TAT derived sequence isTAT(s2-91) (SEQ ID NO: 116).
 7. The transporter cargo conjugate moleculeof claim 2, wherein the sequence is conjugated to any of the traffickingsequences listed having at least 80% or 85%, or at least 90%, or atleast 95%, or at least 99% sequence homology over the whole length ofany of the trafficking sequences of claim
 2. 8. The transporter cargoconjugate molecule of claim 1, wherein the effector molecule is selectedfrom the group consisting of therapeutically active proteins andpeptides, protein kinase inhibitors, inhibitors of protein kinase c-Junamino terminal kinase, antigens, antibodies, apoptotic factors,proteases implicated in pathological states, peptidic proteaseinhibitors, BH3-domains or BH3-only proteins, nucleic acids encodingthese proteins, siRNA, antisense RNAs, cytotoxic agents, and smallorganic compounds.
 9. The transporter cargo conjugate molecule of claim1 further comprising at least one additional component (C), (D) and/or(E) different to component (B), wherein the at least one optionaladditional component (C), (D) and/or (E) is selected independently fromeach other from different effector molecules or their fragments orvariants as defined for component (B).
 10. The transporter cargoconjugate molecule of claim 9, wherein components (A) and (B), andoptionally (C), (D) and/or (E) are covalently linked with each other.11. The transporter cargo conjugate molecule of claim 10, wherein the atleast one additional component (C), (D) and/or (E) is selected from asignal sequence or localization sequence, which directs the inventivetransporter cargo conjugate molecule to a particular intracellulartarget localization or to a particular cell type.
 12. The transportercargo conjugate molecule of claim 11, wherein component (B) and/or theat least one additional component (C), (D) and/or (E) is (are) a proteinor peptide sequence and is (are) composed of L-amino acids, D-aminoacids or a mixture of both.
 13. The transporter cargo conjugate moleculeof claim 12, wherein component (A) is positioned at the C-terminal endof the transporter cargo conjugate molecule, provided that component (B)is a protein or peptide sequence.
 14. A pharmaceutical compositioncomprising a transporter cargo conjugate molecule of claim 1 andoptionally a pharmaceutically acceptable carrier and/or vehicle.
 15. Amethod of prophylaxis, treatment and/or amelioration of a disease, themethod comprising administering to a patient in need thereof atransporter cargo conjugate molecule of claim 1, wherein the disease isselected from the group consisting of cancer or tumor diseases, diseasescaused by defective apoptosis, inflammatory diseases, infectiousdiseases, viral (infectious) diseases, diseases strongly related to JNKsignaling, autoimmune disorders or diseases, cardiovascular diseases,neuronal or neurodegenerative diseases, diseases of the liver, diseasesof the spine, diseases of the uterus, major depressive disorders,non-chronic or chronic inflammatory digestive diseases, hearing loss,diseases of the inner ear.